AU2018207281B2

AU2018207281B2 - Modulating expression of polypeptides via new gene switch expression systems

Info

Publication number: AU2018207281B2
Application number: AU2018207281A
Authority: AU
Inventors: Cheryl G. BOLINGER; Thomas D. Reed; Rutul R. SHAH
Original assignee: Precigen Inc
Current assignee: Precigen Inc
Priority date: 2017-01-10
Filing date: 2018-01-10
Publication date: 2024-08-01
Anticipated expiration: 2038-01-10
Also published as: KR20240128814A; JP2025170778A; KR20190102259A; AU2018207281A1; WO2018132494A1; CN110462040A; EP4382913A2; EP3568474A1; JP2020513777A; JP2025134703A; JP7288401B2; IL310711B1; CN118406655A; US20240263183A1; US11946054B2; SG11201906213UA; CA3049652A1; US20200283778A1; ZA202306868B; JP2023065438A

Abstract

Disclosed herein are polynucleotides encoding ligand-inducible gene switch polypeptides, and systems comprising gene switch polypeptides for modulating the expression of a heterologous gene and an interleukin in a host cell. The compositions, methods and systems described herein facilitate ligand dependent expression of polypeptides including but not limited to cytokines and antigen binding polypeptides.

Description

MODULATING EXPRESSION OF POLYPEPTIDES VIA NEW GENE SWITCH EXPRESSION SYSTEMS

1. CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of U.S. provisional Patent Application Nos. 62/444,775 filed January 10, 2017 and 62/464,958 filed February 28, 2017, which are hereby incorporated by reference in their entirety.

2. REFERENCE TO SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 9, 2018, is named 50471_706_601_SL.txt and is 421,163 bytes in size.

3. INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

4. BACKGROUND OF THE DISCLOSURE

[0004] Adoptive T cell immunotherapy using chimeric antigen receptors (CAR) and T-cell receptors (TCR) has been shown to successfully direct killing of tumor cells. While this innovative technology is promising, the administration of modified T cells into tumor bearing individuals has not been without safety issues, for instance tumor lysis and cytokine release syndrome. In addition, expressing CAR or TCR alone may not be sufficient for efficacy and additional expression of cytokines such as IL-2, IL-12, IL-15 or IL-21 may be needed to increase the efficacy of such treatments. However, this may lead to additional safety issues. It is therefore of interest to gain full control over expression of the therapeutic gene(s) of interest following patient administration.

5. SUMMARY OF THE DISCLOSURE

[0005] Definitions of the specific embodiments of the invention as claimed herein follow

[0005a] In a first aspect, the invention relates to a method for integrating a transgene into the genome of an isolated cell ex vivo, the method comprising delivering to the cell ex vivo: (a) a non-naturally occurring polynucleotide comprising (i) an AttP or AttB recombination sequence, and (ii) a transgene encoding a chimeric antigen receptor or a T-cell receptor; and (b) a polynucleotide encoding an SF370, SP c2, Bxbl, Al18, or >Rvl serine recombinase.

[0005b] Disclosed herein, in certain embodiments, are systems, methods or compositions comprising gene switch polypeptides and polynucleotides encoding the same.

[Text continues on page 2.]

- la-

[0006] Provided herein is a composition that comprises a polynucleotide encoding gene switch polypeptides for ligand-inducible control of heterologous gene expression, wherein said gene switch polypeptides comprise: (a) a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain; and (b) a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain, wherein said first gene switch polypeptide and said second gene switch polypeptide are connected by a linker.

[0007] Provided herein is a composition that comprises one or more polynucleotides encoding a gene switch system for ligand-inducible control of heterologous gene expression, wherein said gene switch system comprises: (a) a first gene switch polypeptide that comprises a transactivation domain; (b)a second gene switch polypeptide that comprises a DNA binding domain fused to a ligand binding domain; and (c) at least one heterologous gene polypeptide; wherein one of said first gene switch polypeptide, said second gene switch polypeptide and said heterologous gene polypeptide is connected by a polypeptide linker to another one of said first gene switch polypeptide, said second gene switch polypeptide and said heterologous gene polypeptide; and wherein said polypeptide linker comprises a cleavable linker or ribosome skipping linker sequence.

[0008] In some embodiments, the DNA binding domain of any of the compositions as provided herein comprises at least one of GAL4 (GAL4 DBD), a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, and a yeast DBD. In some embodiments, the DNA binding domain of any of the compositions as provided herein has a sequence as shown in SEQ ID NO: 184. In some embodiments, the transactivation domain of any of the compositions as provided herein comprises at least one of a VP16 transactivation domain and a B42 acidic activator transactivation domain. Insome embodiments, the transactivation domain of any of the compositions as provided herein has a sequence as shown in SEQ ID NO: 181.

[0009] In some cases, at least one of the first nuclear receptor ligand binding domain, the second nuclear receptor ligand binding domain, and the ligand binding domain of any of the compositions as provided herein comprises at least one of an ecdysone receptor (EcR), a ubiquitous receptor, an orphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor , a steroid hormone receptor like protein, a liver X receptor, a liver X receptor a, a farnesoid X receptor, a receptor interacting protein 14, and a famesol receptor. In other cases, at least one of the first nuclear receptor ligand binding domain, the second nuclear receptor ligand binding domain, and the ligand binding domain of any of the compositions as provided herein has a sequence as shown in any one of

SEQ ID NOs: 185-186. In another case, the first gene switch polypeptide comprises a GAL4 DBD fused to an EcR nuclear receptor ligand binding domain, and said second gene switch polypeptide comprises a VP16 transactivation domain fused to a retinoid receptor X (RXR) nuclear receptor ligand binding domain. In yet another case, the Gal4 DBD fused to the EcR nuclear receptor ligand binding domain has a sequence as shown in any one of SEQ ID NOs: 185-186 or 187-188, and the VP16 transactivation domain fused to the retinoid receptor X (RXR) nuclear receptor ligand binding domain has a sequence as shown in SEQ ID NO: 183.

[0010] In some cases, the linker of any of the compositions as provided herein is a cleavable linker, a ribosome skipping linker sequence or an RES linker. In some cases, the linker is an IRES linker and has a sequence as shown in any one of SEQ ID NOs: 18-19. In other cases, the linker is a cleavable linker or a ribosome skipping linker sequence. In some embodiments, the cleavable linker or the ribosome skipping linker sequence comprises one or more of a 2A linker, p2A linker, T2A linker, F2A linker, E2A linker, GSG-2A linker, GSG linker, SGSG linker, furinlink linker variants and derivatives thereof. In other embodiments, the cleavable linker or said ribosome skipping linker sequence has a sequence as shown in any one of SEQ ID NOs: 146-162.

[0011] In an embodiment, the polynucleotide or the one or more polynucleotides of any of the compositions as provided herein further encodes an antigen-binding polypeptide. In another embodiment, the antigen-binding polypeptide of any of the compositions as provided herein comprises at least one of a chimeric antigen receptor (CAR) and a T-cell receptor. In other embodiments, the antigen-binding polypeptide comprises a CAR and said CAR is capable of binding at least one of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-Al,, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2. In another embodiment, the antigen-binding polypeptide comprises a CAR and the CAR has a sequence as shown in any one of SEQ ID NOs: 210-244.

[0012] In other embodiments, the polynucleotide or the one or more polynucleotides of any of the compositions as provided herein further encodes a cell tag. In some cases, the cell tag comprises at least one of a HERI truncated variant or a CD20 truncated variant. In other cases, the cell tag has a sequence as shown in any one of SEQ ID NOs: 189-202.

[0013] In some embodiments, expression of at least one of the first gene switch polypeptide, the second gene switch polypeptide, the antigen-binding polypeptide, and the cell tag of any of the compositions as provided herein is modulated by a promoter, wherein the promoter is a tissue-specific promoter or an EF1A promoter or functional variant thereof. Insomecases,the promoter is an EF1A promoter or functional variant thereof having a sequence as shown in any one of SEQ ID NOs: 58-60. In other cases, the promoter is a tissue-specific promoter comprising a T-cell-specific response element. In another case, the tissue-specific promoter comprises one or more NFAT response element(s). In yet another case, the NFAT response element has a sequence as shown in any one of SEQ ID NOs: 51-57.

[0014] In some examples, any of the compositions as provided herein further comprises a second polynucleotide encoding a heterologous gene polypeptide. In some cases, the heterologous gene polypeptide comprises at least one of a cytokine, a cell tag, and a chimeric antigen receptor (CAR). In some cases, the cytokine comprises a cytokine and said cytokine comprises at least one of IL-1, IL-2, IL-15, IL-12, IL-21, a fusion of IL-15, IL-15RU, or an IL 15 variant. In other cases, the cytokine is in secreted form. In another case, the cytokine is in membrane-bound form. In yet another case, the cytokine has a sequence as shown in any one of SEQ ID NOs: 203-209.

[0015] In an embodiment, expression of the at least one heterologous gene polypeptide of any of the compositions as provided herein is modulated by an inducible promoter. In some embodiments, the inducible promoter has a sequence as shown in any of SEQ ID NOs: 40-64. In other embodiments, the inducible promoter is modulated by at least one of the first gene switch polypeptide and the second gene switch polypeptide.

[0016] Also provided herein is a vector that comprises the polynucleotide(s) of any of the compositions as provided herein. In an embodiment, the vector is a lentivirus vector, a retroviral vector, or a non-viral vector. In another embodiment, the non-viral vector is a Sleeping Beauty transposon.

[0017] Provided herein is a method of regulating the expression of a heterologous gene in an effector cell, the method comprising: (a) introducing into said effector cell one or more polynucleotides that encode (i) a first gene switch polypeptide that comprises a DNA-binding domain fused to a ligand binding domain, (ii) a second gene switch polypeptide that comprises a transactivation domain, (iii) a heterologous gene polypeptide encoded by said heterologous gene and (iv) a polypeptide linker that comprises a cleavable or ribosome skipping linker sequence, wherein said polypeptide linker connects one of said first gene switch polypeptide, said second gene switch polypeptide and said heterologous gene polypeptide to another one of said first gene switch polypeptide, said second gene switch polypeptide and said heterologous gene polypeptide; and (b) contacting said effector cell with a ligand in an amount sufficient to induce expression of said heterologous gene.

[0018] In an example, at least one of the one or more polynucleotides of the method of regulating the expression of the heterologous gene in the effector cell as provided herein further encodes an antigen-binding polypeptide. In some cases, the antigen-binding polypeptide selectively binds a predetermined cell surface protein of a target cell. In some cases, the target cell is a mammalian cell. In other cases, the target cell is a tumor cell. In some embodiments, the predetermined cell surface protein of the method of regulating the expression of the heterologous gene in the effector cell as provided herein is a tumor antigen. In some cases, the antigen-binding polypeptide selectively binds the predetermined cell surface protein of the target cell prior to contacting the effector cell with the ligand. In some cases, the effector cell is exposed to the predetermined cell surface protein of the target cell for at least 7 days prior to contacting the effector cell with the ligand. In other cases, binding of the predetermined cell surface protein by the antigen-binding polypeptide activates the effector cell. In some embodiments, the method further comprises co-culturing said effector cell with an artificial antigen presenting cell (aAPC) expressing said predetermined cell surface protein, wherein binding of said antigen-binding polypeptide to said predetermined cell surface protein of said aAPC activates said effector cell. In some embodiments, the co-culturing is for at least 7 days, 14 days, 21 days or 28 days. In some cases, the aAPC is a transgenic K562 cell.

[0019] In other embodiments, the antigen-binding polypeptide in the method of regulating the expression of the heterologous gene in the effector cell as provided herein comprises at least one of a chimeric antigen receptor (CAR) and a T-cell receptor. In another embodiment, the antigen binding polypeptide comprises a CAR and said CAR is capable of binding at least one of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A, MUC-16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2. In yet another embodiment, the antigen-binding polypeptide comprises a CAR having a sequence as shown in any one of SEQ ID NOs 210-244. In some embodiments, the heterologous gene polypeptide comprises the antigen-binding polypeptide.

[0020] In some cases, at least one of the one or more polynucleotides in the method of regulating the expression of the heterologous gene in the effector cell as provided herein further encodes a cell tag. In some cases, the cell tag comprises at least one of a HER truncated variant and a CD20 truncated variant. In other cases, the cell tag has a sequence as shown in any one of SEQ ID NOs: 189-202. In some cases, the heterologous gene polypeptide comprises the cell tag.

[0021] In other cases, at least one of the one or more polynucleotides in the method of regulating the expression of the heterologous gene in the effector cell as provided herein further encodes a cytokine. In some cases, the cytokine comprises at least one of IL-1, IL-2, IL-15, IL

12, IL-21, a fusion of IL-15, IL-15Ra, or an IL-15 variant. In an embodiment, the cytokine is in secreted form. In another embodiment, the cytokine is in membrane-bound form. In yet another embodiment, the cytokine has a sequence as shown in any one of SEQ ID NOs: 203-209. In other embodiments, the heterologous gene polypeptide comprises said cytokine.

[0022] In an embodiment, expression of the heterologous gene polypeptide in the method of regulating the expression of the heterologous gene in the effector cell as provided herein is modulated by an inducible promoter. In certain embodiments, the inducible promoter has a sequence as shown in any one of SEQ ID NOs: 40-64.

[0023] In another embodiment, the DNA-binding domain in the method of regulating the expression of the heterologous gene in the effector cell as provided herein comprises at least one of GAL4 (GAL4 DBD), a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, and a yeast DBD. In yet another embodiment, the DNA binding domain has a sequence as shown in SEQ ID NO: 184.

[0024] In yet another embodiment, the transactivation domain in the method of regulating the expression of the heterologous gene in the effector cell as provided herein comprises at least one of a VP16 transactivation domain, and a B42 acidic activator transactivation domain. In some cases, the transactivation domain has a sequence as shown in SEQ ID NO: 181. In other embodiments, at least one of said first and second gene switch polypeptides further comprises a response element capable of binding to said DNA-binding domain.

[0025] In some embodiments, the ligand binding domain in the method of regulating the expression of the heterologous gene in the effector cell as provided herein comprises at least one of an ecdysone receptor (EcR), a ubiquitous receptor, an orphan receptor 1, an NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor p, a steroid hormone receptor like protein, a liver X receptor, a liver X receptor a, a farnesoid X receptor, a receptor interacting protein 14, and a famesol receptor. In certain embodiments, the ligand binding domain has a sequence as shown in any one of SEQ ID NOs: 185-186. In certain embodiments, the first gene switch polypeptide comprises a GAL4 DBD fused to an EcR nuclear receptor ligand binding domain, and the second gene switch polypeptide comprises a VP16 transactivation domain fused to a retinoid receptor X (RXR) nuclear receptor ligand binding domain. In some embodiments, the Gal4 DBD fused to an EcR has a sequence as shown in any one of SEQ ID NOs: 185-186 and said VP16 transactivation domain fused to a retinoid receptor X (RXR) nuclear receptor ligand binding domain has a sequence as shown in SEQ ID NO: 183.

[0026] In certain embodiments, the ligand in the method of regulating the expression of the heterologous gene in the effector cell as provided herein comprises at least one of: (2S,3R,5R,9R,1OR,13R,14S,17R)-17- [(2S,3R)-3,6-dihydroxy-6-methylheptan- 2-yl]-2,3,14 trihydroxy-10,13-dimethyl- 2,3,4,5,9,11,12,15,16,17-decahydro- 1H-cyclopenta[a]phenanthren 6-one; N'-(3,5-Dimethylbenzoyl)-N'-[(3R)-2,2-dimethyl-3-hexanyl]-2-ethyl-3 methoxybenzohydrazide; 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5 dimethyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-Methyl-2,3-dihydro benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2 dimethyl-propyl)-hydrazide; 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1 tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl)-hydrazide; 5-Methyl-2,3-dihydro-benzo[1,4]dioxine 6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide; 5 Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethyl-benzoyl)-N'-(1-ethyl 2,2-dimethyl-propyl)-hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N' (3,5-dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-Ethyl-2,3 dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl) hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(-tert-butyl-butyl)-N' (3,5-dimethoxy-4-methyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2 dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5 Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5 Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methoxy-2-methyl-benzoyl) hydrazide; 3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-ethyl-3-methoxy benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N' (2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2 ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 2-Methoxy-nicotinic acid N-(1-tert-butyl pentyl)-N'-(4-ethyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(2,2-dimethyl-1-phenyl propyl)-N'-(4-ethyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-pentyl)-N' (3-methoxy-2-methyl-benzoyl)-hydrazide; and 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide.

[0027] In some cases, the expression of the heterologous gene in the method of regulating the expression of the heterologous gene in the effector cell as provided herein is reduced or eliminated in the absence of the ligand, as compared to the expression in the presence of the ligand. In certain cases, the expression of said heterologous gene is restored by providing additional amounts of the ligand.

[0028] In other cases, expression of at least one of the first gene switch polypeptide and the second gene switch polypeptide is modulated by a promoter in the method of regulating the expression of the heterologous gene in the effector cell as provided herein, wherein said promoter is a tissue-specific promoter or an EF1A promoter or functional variant thereof. Incertaincases, the promoter is an EF1A promoter or functional variant thereof having a sequence as shown in any one of SEQ ID NOs: 58-60. In other cases, the promoter is a tissue-specific promoter, and the tissue-specific promoter comprises a T-cell-specific response element. In another case, the tissue-specific promoter comprises one or more NFAT response element(s). In yet another case, the NFAT response element has a sequence as shown in any one of SEQ ID NOs: 50-57.

[0029] In certain cases, the one or more polynucleotides of the method of regulating the expression of the heterologous gene in the effector cell as provided herein are comprised within a vector. In some cases, the vector is a lentivirus vector, a retroviral vector, or a non-viral vector. In certain cases, the non-viral vector is a Sleeping Beauty transposon.

[0030] Provided herein is a gene switch system for ligand-inducible control of heterologous gene expression, wherein said gene switch system comprises one or more expression cassettes, wherein said one or more expression cassettes comprise: (a) a sequence encoding a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain; and (b) a sequence encoding a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain; wherein said first gene switch polypeptide and said second gene switch polypeptide are connected by a polypeptide linker.

[0031] Provided herein is a gene switch system for ligand-inducible control of heterologous gene expression, wherein said gene switch system comprises one or more expression cassettes, wherein said one or more expression cassettes comprise: (a) a sequence encoding a first gene switch polypeptide that comprises a transactivation domain; (b) a sequence encoding a second gene switch polypeptide that comprises a DNA binding domain fused to a ligand binding domain; and (c) a sequence encoding a heterologous gene polypeptide; wherein one of said transactivation domain, DNA binding domain fused to said ligand binding domain, and said heterologous gene polypeptide is connected by a polypeptide linker to another of said transactivation domain, DNA binding domain fused to said ligand binding domain, and said heterologous gene polypeptide, and wherein said polypeptide linker comprises a cleavable or ribosome skipping linker sequence.

[0032] In some embodiments, the one or more expression cassettes of the gene switch system as provided herein further comprise a sequence encoding a heterologous gene polypeptide. In some embodiments, the one or more expression cassettes further comprise one or more of the following: (a) one or more recombinase attachment sites; and (b) a sequence encoding a serine recombinase. In other embodiments, the one or more expression cassettes further comprise one or more of the following: (a) a non-inducible promoter; and (b) an inducible promoter.

[0033] In certain embodiments, the DNA binding domain of the gene switch system as provided herein has a sequence as shown in SEQ ID NO: 184. In other embodiments, the transactivation domain of the gene switch system as provided herein has a sequence as shown in SEQ ID NO: 181. In another embodiment, at least one of the first and second nuclear receptor ligand binding domains and the ligand binding domain has a sequence as shown in any one of SEQ ID NOs: 185-186. In certain embodiments, the non-inducible promoter has a sequence as shown in any one of SEQ ID NOs: 40-64. In certain embodiments, the inducible promoter has a sequence as shown in any one of SEQ ID NOs: 40-64. In certain embodiments, the polypeptide linker has a sequence as shown in any one of SEQ ID NOs: 146-162. In some embodiments, the polypeptide linker is a cleavable linker, a ribosome skipping linker or an IRES linker with a sequence as shown in any one of SEQ ID NOs: 18-19 and 146-162.

[0034] In some examples, the one or more expression cassettes of the gene switch system as provided herein include an expression cassette that comprises a sequence encoding a chimeric antigen receptor (CAR), wherein expression of the chimeric antigen receptor is modulated by a non-inducible promoter. In certain examples, the CAR is capable of binding at least one of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-Al, MUC-16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF R2.

[0035] In other examples, the non-inducible promoter of the gene switch system as provided herein comprises EF1A or a variant thereof In certain cases, the expression cassette has a sequence as shown in SEQ ID NO: 131. In other cases, the expression cassette further comprises a sequence encoding a cell tag, wherein the cell tag is connected to the CAR by a linker. In some cases, the cell tag comprises at least one of a HER1 truncated variant and a CD20 truncated variant.

[0036] In another example, the expression cassette of the gene switch system as provided herein has a sequence as shown in SEQ ID NO: 132. In some embodiments, the expression cassette further comprises a sequence encoding the first gene switch polypeptide and a sequence encoding the second gene switch polypeptide, wherein one of the first and second gene switch polypeptides is connected to the CAR by a linker.

[0037] In some embodiments, the first and second gene switch polypeptides of the gene switch system as provided herein are connected by said polypeptide linker, and the polypeptide linker is a cleavable linker. In some embodiments, the expression cassette has a sequence as shown in SEQ ID NO: 133. In some embodiments, the first and second gene switch polypeptides are connected by the polypeptide linker, and the polypeptide linker is an IRES linker.

[0038] In certain embodiments, the expression cassette of the gene switch system as provided herein has a sequence as shown in SEQ ID NO: 134. In other embodiments, the one or more expression cassettes includes an expression cassette that comprises a sequence encoding the heterologous gene polypeptide, wherein expression of said heterologous gene polypeptide is modulated by an inducible promoter. In other embodiments, the heterologous gene polypeptide comprises acytokine. In some cases, the cytokine comprises at least one of IL-1, IL-2, IL-15, IL-12, IL-21, a fusion of IL-15, IL-15Ra, or an IL-15 variant.

[0039] In another embodiment, the expression cassette of the gene switch system as provided herein has a sequence as shown in SEQ ID NO: 135. In some embodiments, the expression cassette comprises a sequence encoding a second heterologous gene polypeptide, wherein the second heterologous gene polypeptide comprises a cell tag. In certain cases, the cell tag comprises at least one of a HERI truncated variant and a CD20 truncated variant. In other cases, the cell tag is connected to the cytokine by a linker. In another case, the expression cassette has a sequence as shown in SEQ ID NO: 136.

[0040] In some cases, the gene switch system as provided herein is for integrating a heterologous gene in a host cell, wherein upon contacting the host cell with the one or more expression cassettes in the presence of the seine recombinase and the one or more recombinase attachment sites, the heterologous gene is integrated in the host cell. In certain cases, the gene switch system further comprises a ligand, wherein the heterologous gene is expressed in the host cell upon contacting the host cell with the ligand. In certain cases, the host cell is a T cell or an NK cell. In certain cases, the one or more recombinase attachment sites can comprise a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB). In some cases, the seine recombinase can be SF370.

[0041] In certain cases, the inducible promoter of the gene switch system as provided herein is activated by the transactivation domain. In certain cases, the system is contained in one or more vectors. In certain cases, the system is contained in one vector.

[0042] Disclosed is a polynucleotide encoding one or more of the components of the gene switch system as provided herein. Also disclosed herein is a vector that comprises the polynucleotide encoding one or more of the components of the gene switch system as provided herein. In certain cases, the vector is any one of a lentivirus vector, a retroviral vector, or a non viral vector. In certain cases, the vector is a non-viral vector and said non-viral vector is a Sleeping Beauty transposon.

[0043] Provided herein is a polynucleotide construct that comprises: a polynucleotide that encodes a first gene switch polypeptide, a polynucleotide that encodes a second gene switch polypeptide, and a polynucleotide that encodes a gene of interest (GOI), wherein the polynucleotide that encodes the GOI comprises a continuous open-reading frame (ORF) positioned between the polynucleotide that encodes the first gene switch polypeptide and the polynucleotide that encodes the second gene switch polypeptide, wherein the polynucleotide construct further comprises a polynucleotide that encodes a linker, and wherein said GOI is connected by said linker to each of said first and second gene switch polypeptides.

[0044] Provided herein is a polynucleotide that comprises at least one of: (i) a first sequence that encodes a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain; and (ii) a second sequence that encodes a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain; wherein expression of at least one of said first sequence and said second sequence is modulated by one or more NFAT response element.

[0045] Provided herein is a-method of stimulating the proliferation and/or survival of engineered cells, the method comprising: (a) obtaining a sample of cells from a subject, and (b) transfecting cells of said sample of cells with one or more polynucleotides that comprise one or more transposons, wherein said one or more transposons encode: a chimeric antigen receptor (CAR) or a TCR, a cytokine, one or more cell tags, gene switch polypeptides for ligand-inducible control of said cytokine and a transposase effective to integrate said one or more polynucleotides into the genome of said cells, to provide a population of engineered cells; wherein said gene switch polypeptides comprise: i) a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain, wherein said first gene switch polypeptide and said second gene switch polypeptide are connected by a linker.

[0046] Provided herein is a method of enhancing in vivo persistence of engineered cells in a subject in need thereof, the method comprising: (a) obtaining a sample of cells from a subject, and (b) transfecting cells of said sample of cells with one or more polynucleotides that comprise one or more transposons, wherein said one or more transposons encode: a chimeric antigen receptor (CAR) or a TCR, a cytokine, one or more cell tags, gene switch polypeptides for ligand inducible control of said cytokine and a transposase effective to integrate the DNA into the genome of said cells, to provide a population of engineered cells; wherein said gene switch polypeptides comprise: i) a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain, wherein said first gene switch polypeptide and said second gene switch polypeptide are connected by a linker.

[0047] In some embodiments, the transfecting cells in the methods provided herein comprises electroporating the cells. In certain embodiments, at least one polynucleotide of the one or more polynucleotides encodes the gene switch polypeptides, and the at least one polynucleotide is modulated by a promoter, wherein the promoter is a tissue-specific promoter or an EF1A promoter or functional variant thereof. In certain cases, the promoter is an EF1A promoter or functional variant thereof having a sequence as shown in any one of SEQ ID NOs: 58-60. In certain cases, the promoter is a tissue-specific promoter, and the tissue-specific promoter comprises a T-cell-specific response element. In certain cases, the promoter is a tissue-specific promoter, and the tissue-specific promoter comprises one or more NFAT response element. In certain cases, the NFAT response element has a sequence as shown in any one of SEQ ID NOs: 50-57. In certain cases, the cytokine comprises at least one of IL-1, IL-2, IL-15, IL-12, IL-21, a fusion of IL-15, IL-15Ra, or an IL-15 variant. In other cases, the cytokine is in secreted form. In another case, the cytokine is in membrane-bound form. In yet another case, the cytokine has a sequence as shown in any one of SEQ ID NOs: 203-209. In yet another case, the cells are NK cells, NKT cells, T-cells or T-cell progenitor cells.

[0048] In other cases, the method as provided herein further comprises administering an effective amount of the engineered cells to a subject in need thereof. In certain cases, administering comprises immediately infusing the subject with the engineered cells. In certain cases, the method further comprises administering an effective amount of the ligand to induce expression of the cytokine. In certain cases, the ligand is veledimex. In some cases, the CAR is capable of binding at least one of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-Al, MUC-16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2. In certain cases, the CAR is capable of binding CD19. In certain cases, the CAR is capable of binding CD33. In certain cases, the transposase is salmonid type Tcl-like transposase. In certain cases, the transposase is SB11 or SB100x transposase. In certain cases, the one or more cell tags comprises at least one of a HERI truncated variant and a CD20 truncated variant. In certain cases, the one or more cell tags has a sequence as shown in any one of SEQ ID NOs: 189-202.

[0049] Provided herein is a method of treating a subject with a solid tumor, the method comprising: (a) obtaining a sample of cells from a subject, (b) transfecting cells of said sample of cells with one or more polynucleotides that comprise one or more transposons, wherein said one or more transposons encode: a chimeric antigen receptor (CAR) or a TCR, a cytokine, one or more cell tags, gene switch polypeptides for ligand-inducible control of said cytokine and a transposase effective to integrate the DNA into the genome of said cells, to provide a population of engineered cells; wherein said gene switch polypeptides comprise: i) a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain, wherein said first gene switch polypeptide and said second gene switch polypeptide are connected by a linker, and (c) administering said population of engineered cells to said subject.

[0050] In some embodiments, the cytokine in the method of treating comprises at least one of IL-1, IL-2, IL-15, IL-12, IL-21, a fusion of IL-15, IL-15Ra, or an IL-15 variant. In some embodiments, the cytokine comprises IL-12. In some embodiments, at least one polynucleotide of the one or more polynucleotides encodes the gene switch polypeptides, and the at least one polynucleotide is modulated by a promoter, wherein the promoter is a tissue-specific promoter or an EF1A promoter or functional variant thereof. In some embodiments, the promoter is an EF1A promoter or functional variant thereof having a sequence as shown in any one of SEQ ID NOs: 58-60. In certain embodiments, the promoter is a tissue-specific promoter, and the tissue-specific promoter comprises a T-cell-specific response element. In some embodiments, the promoter is a tissue-specific promoter, and the tissue-specific promoter comprises one or more NFAT response element. In some embodiments, the NFAT response element has a sequence as shown in any one of SEQ ID NOs: 50-57.

[0051] Also disclosed is an engineered effector cell, wherein said engineered effector cell comprises: (a) a first polynucleotide encoding an engineered receptor construct that selectively binds a predetermined cell surface protein expressed by a target cell; (b) one or more polynucleotides encoding one or more engineered gene switch polypeptides, wherein said engineered gene switch polypeptides comprise one or more of a transactivation domain, a DNA binding domain, and a ligand binding domain; and (c) a heterologous gene under the control of a ligand-inducible promoter modulated by said engineered gene switch polypeptides; wherein said heterologous gene encodes a cytokine.

[0052] In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a tumor cell. In some embodiments, the cell surface protein is expressed on the surface of said tumor cell. In some embodiments, the cell surface protein is expressed in an artificial antigen-presenting cell (aAPC). In some embodiments, the engineered receptor construct is a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor binds CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP 40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-Al, h5T4, PSMA, TAG-72, EGFRvIII, CD123 or VEGF-R2. In some embodiments, the chimeric antigen receptor binds EGFRvIII.

[0053] In some embodiments, the engineered receptor construct is an engineered T-cell receptor. In some embodiments, the cytokine is IL-2, IL-15, IL-12, IL-21, or a fusion of IL-15 and IL-15Ra. In some embodiments, the cytokine is IL-12. In some embodiments, the effector cell further comprises a polynucleotide encoding a protein comprising a cell tag. In some embodiments, the cell tag is a truncated HERI variant or a CD20 truncated variant. In some embodiments, the effector cell is an immune effector cell. In some embodiments, the effector cell is a T cell, an NK cell or a tumor infiltrating lymphocyte. In some embodiments, the one or more engineered gene switch polypeptides comprise a transactivation domain, a DNA binding domain and a ligand binding domain. In some embodiments, the one or more engineered gene switch polypeptides comprise a first gene switch polypeptide comprising said DNA-binding domain fused to a nuclear receptor ligand binding domain and a second gene switch polypeptide comprising said transactivation domain fused to a nuclear receptor ligand binding domain.

[0054] In some embodiments, the first gene switch polypeptide and said second gene switch polypeptide are connected by a polypeptide linker. In some embodiments, the polypeptide linker is a cleavable or ribosome skipping linker sequence. In some embodiments, the polypeptide linker comprises one of 2A, F/T2A, GSG-2A, GSG linker, SGSG linker, furinlink variants and derivatives thereof. In some embodiments, the transactivation domain comprises a VP16 transactivation domain. In some embodiments, the one or more engineered gene switch polypeptides comprise at least one of an ecdysone receptor (EcR), a ubiquitous receptor, an orphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor , a steroid hormone receptor like protein, a liver X receptor, a liver X receptor a, a farnesoid X receptor, a receptor interacting protein 14, and a famesol receptor. In some embodiments, the DNA-binding domain (DBD) comprises at least one of GAL4 (GAL4 DBD), a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, and a yeast DBD.

[0055] Further disclosed herein is a method of modulating the expression of a heterologous gene, the method comprising contacting the target cell with an effector cell disclosed herein. In some cases, the modulating comprises increasing or decreasing expression of said heterologous gene. In some cases, the expression is reduced or eliminated in the absence of said ligand, as compared to the expression in the presence of said ligand. In some cases, the expression is resuscitated by providing additional amounts of said ligand.

[0056] Further disclosed is a method of regulating the expression of a heterologous in an effector cell, the method comprising: introducing into said effector cell polynucleotides encoding one or more engineered gene switch polypeptides, an engineered receptor construct, and said heterologous gene; wherein said heterologous gene is under the control of an inducible promoter; activating said effector cell via said engineered receptor construct; and presenting a ligand to said effector cell for inducing expression of said heterologous gene via said one or more engineered gene switch polypeptides after said activating said effector cell.

[0057] In some cases, the activating comprises contacting the effector cell with an antigen. In some cases, the antigen is a tumor antigen. In some cases, the effector cell is exposed to the tumor antigen for at least 7 days prior to presenting a ligand to the effector cell. In some cases, activating the effector cell comprises co-culturing the effector cell with an artificial antigen presenting cell (aAPC). In some cases, the co-culturing is for at least 7 days, 14 days, 21 days or 28 days. In some cases, the aAPC is a transgenic K562 cell. In some cases, the engineered receptor construct comprises a chimeric antigen receptor (CAR). In some cases, the CAR binds CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-Al, h5T4, PSMA, TAG-72, EGFRvIII, CD123, or VEGF-R2. In some cases, the engineered receptor construct comprises an engineered T-cell receptor (TCR). In some cases, the effector cell comprises contacting the effector cell with a TCR-binding polypeptide. In some cases, the TCR-binding polypeptide comprises a TCR-binding antibody or fragment thereof. In some cases, the effector cell is exposed to the TCR-binding polypeptide for at least 7 days prior to presenting a ligand to the effector cell. In some cases, the TCR-binding polypeptide is expressed by an aAPC. In some cases, the heterologous gene encodes a cytokine. In some cases, the cytokine is IL-2, IL-15, IL-12, IL-21, or a fusion of IL-15 and IL-15RU. In some cases, the cytokine is IL-12.

[0058] In some cases, the heterologous gene encodes at least one cell tag. In some cases, the cell tag is a HERI truncated variant or a CD20 truncated variant. In some cases, the one or more gene expression cassettes further comprise a nucleotide sequence encoding a cell tag. In some cases, the effector cell is an immune effector cell. In some cases, the immune effector cell is a T cell, an NK cell or a tumor infiltrating lymphocyte. In some cases, the one or more engineered gene switch polypeptides comprise a transactivation domain, a DNA-binding domain, and a ligand-binding domain. In some cases, the ligand-binding domain comprises at least one of an ecdysone receptor (EcR), a ubiquitous receptor, an orphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor p, a steroid hormone receptor like protein, a liver X receptor, a liver X receptor a, a farnesoid X receptor, a receptor interacting protein 14, and a farnesol receptor. In some cases, the DNA binding domain comprises at least one of GAL4 (GAL4 DBD), a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, and a yeast DBD. In some cases, the transactivation domain comprises a VP16 transactivation domain.

[0059] In some cases, the one or more engineered gene switch polypeptides further comprise a response element capable of binding to said DNA-binding domain. In some cases, the one or more engineered gene switch polypeptides further comprise at least one of ultraspiracle protein (USP), retinoid receptor X (RXR), or functional fragments and variants thereof, wherein said functional fragments and variants thereof are capable of binding to an EcR. In some cases, the one or more engineered gene switch polypeptides further comprise one or more polypeptide linkers. In some cases, the one or more polypeptide linkers further comprise at least one of 2A, GSG-2A, GSG linker, SGSG linker, furinlink variants and derivatives thereof. In some cases, the ligand comprises at least one of. (2S,3R,5R,9R,1OR,13R,14S,17R)-17- [(2S,3R)-3,6-dihydroxy 6-methylheptan- 2-yl]-2,3,14-trihydroxy-10,13-dimethyl- 2,3,4,5,9,11,12,15,16,17-decahydro 1H-cyclopenta[a]phenanthren-6-one; N'-(3,5-Dimethylbenzoyl)-N'-[(3R)-2,2-dimethyl-3 hexanyl]-2-ethyl-3-methoxybenzohydrazide; 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6 carboxylic acid N'-(3,5-dimethyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5 Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy-4-methyl-benzoyl) N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6 carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl)-hydrazide; 5-Methyl-2,3 dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4 methyl-benzoyl)-hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5 dimethyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-Ethyl-2,3-dihydro benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2 dimethyl-propyl)-hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1-tert butyl-butyl)-N'-(3,5-dimethyl-benzoyl)-hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6 carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide; 3,5 Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl) hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(3 methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3 methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl butyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2 dimethyl-propyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethyl benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethoxy 4-methyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy- benzoyl)-hydrazide; 2 Methoxy-nicotinic acid N-(1-tert-butyl-pentyl)-N'-(4-ethyl-benzoyl)-hydrazide; 3,5-Dimethyl benzoic acid N-(2,2-dimethyl-1-phenyl-propyl)-N'-(4-ethyl-benzoyl)-hydrazide; 3,5-Dimethyl benzoic acid N-(1-tert-butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; and 3,5

Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl) hydrazide.

[0060] Further disclosed herein is a polynucleotide for ligand-inducible control of heterologous gene expression in an engineered cell, wherein said polynucleotide comprises: (a) a first sequence encoding an engineered receptor construct capable of binding a predetermined cell surface protein expressed by a target cell; and (b) a second sequence encoding one or more engineered gene switch polypeptides for ligand-inducible control of said heterologous gene expression by modulating a promoter linked to said heterologous gene in response to the presence of said ligand; wherein said engineered receptor construct and said one or more engineered gene switch polypeptides are connected by a polypeptide linker.

[0061] In some embodiments, the polypeptide linker comprises a cleavable linker sequence. In some embodiments, the cleavable linker sequence is an F2A linker. In some embodiments, the one or more engineered gene switch polypeptides comprise a first gene switch polypeptide comprising a DNA-binding domain fused to a nuclear receptor ligand binding domain, and a second gene switch polypeptide comprising a transactivation domain fused to a nuclear receptor ligand binding domain. In some embodiments, the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In some embodiments, the linker is a cleavable or ribosome skipping linker sequence. In some embodiments, the linker is a 2A linker, GSG-2A linker, GSG linker, SGSG linker, furinlink variants and derivatives thereof In some embodiments, the 2A linker is an F2A linker. In some embodiments, the heterologous gene encodes a cytokine. In some embodiments, the cytokine is IL-2, IL-15, IL-12, IL-21, or a fusion of IL-15 and IL-15RU. In some embodiments, the cytokine is IL-12. In some embodiments, the heterologous gene encodes at least one cell tag. In some embodiments, the cell tag is a HERI truncated variant or a CD20 truncated variant. In some embodiments, the polynucleotide is incorporated into an engineered cell.

[0062] Further disclosed herein is a system for expressing a cytokine in a host cell in a vicinity of a target cell, wherein the system comprises: (a) the host cell encoding: (i) an engineered receptor construct that selectively binds a predetermined cell surface protein expressed in the target cell; (ii) an engineered gene switch polypeptide, wherein the engineered gene switch polypeptide comprises one or more of a transactivation domain, a DNA-binding domain, and a ligand-binding domain; and (iii) a cytokine expressed from a heterologous gene linked to a promoter modulated by the engineered gene switch polypeptide; and (b) a ligand; such that upon binding of the engineered receptor construct to the cell surface protein in the presence of the ligand, the cytokine is expressed by the host cell in the vicinity of the target cell.

[0063] In some cases, the engineered receptor construct comprises a chimeric antigen receptor. In some cases, the chimeric antigen receptor binds CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-Al, h5T4, PSMA, TAG-72, EGFRvIII, CD123 or VEGF-R2. In some cases, the chimeric antigen receptor binds EGFRvIII. In some cases, the engineered receptor construct comprises an engineered T-cell receptor. In some cases, the cytokine is IL-2, IL-15, IL-12, IL-21, or a fusion of IL-15 and IL

15Ra. In some cases, the cytokine is IL-12. In some cases, the host cell further encodes at least one cell tag. In some cases, the cell tag is a HER truncated variant or a CD20 truncated variant.

[0064] Further provided for herein is a method of expressing a cytokine from a host cell in the vicinity of a target cell, the method comprising contacting a system disclosed herein the said target cell in the presence of the ligand. Further provided for herein is a method of modulating the expression of a cytokine from a host cell in the vicinity of a target cell, the method comprising contacting a system disclosed herein with the target cell in the presence of the ligand, and regulating the amount of ligand. In some cases, the expression is reduced or eliminated in the absence of said ligand, as compared to expression in the presence of said ligand. In some cases, the expression is resuscitated by providing additional amounts of said ligand. In some cases, the host cell is an animal cell or a mammalian cell. In some cases, the mammalian cell is a human cell. In some cases, the mammalian cell is a T-cell, an NK cell, or a tumor-infiltrating lymphocyte. In some cases, the target cell is a tumor cell. In some cases, the system is contained in one or more vectors. In some cases, each vector comprises a plasmid. In some cases, each vector comprises an expression plasmid. In some cases, each vector is a lentivirus vector, a retroviral vector, or a non-viral vector. In some cases, the non-viral vector comprises a Sleeping Beauty transposase and a SB transposon.

[0065] Further disclosed herein is a polypeptide for binding to EGFRvIII, wherein the polypeptide comprises a polypeptide sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to a sequence shown in SEQ ID NOs: 233, 234, 235, 236, 237, 238, 239,240,241,242,243, or244.

[0066] In some cases, the polypeptide is expressed in an engineered effector cell. In some cases, the effector cell is an immune effector cell. In some cases, the immune effector cell is a T cell, an NK cell or a tumor- infiltrating lymphocyte. In some cases, the polypeptide comprises an antibody or fragment thereof. In some cases, the polypeptide comprises a chimeric antigen receptor (CAR). In some cases, the EGFRvIII binds an scFv antigen-binding domain of said antibody or fragment thereof or said CAR. In some cases, the polypeptide does not cross-react with wild-type EGFR.

[0067] Further disclosed herein is a polynucleotide encoding: a) an engineered receptor construct capable of binding EGFRvIII; b) a first polypeptide comprising a transactivation domain and a nuclear receptor ligand binding domain; and (c) a second polypeptide comprising a DNA-binding domain and a nuclear receptor ligand binding domain; wherein an F/T2A linker connects at least two of the engineered receptor construct, the first polypeptide and the second polypeptide.

[0068] In some cases, the F/T2A linker connects said engineered receptor construct to at least one of said first polypeptide and said second polypeptide. In some cases, the F/T2A linker connects the first polypeptide to said second polypeptide. In some cases, the polynucleotide further encodes an IRES linker. In some cases, the RES linker connects the first polypeptide to the second polypeptide.

[0069] Further disclosed herein is an effector cell comprising a polynucleotide disclosed herein. In some cases, the effector cell encodes a heterologous gene linked to an inducible promoter capable of being modulated by said first polypeptide or said second polypeptide.

[0070] Further disclosed herein is a chimeric antigen receptor (CAR) capable of binding EGFRvIII, wherein the CAR comprises: (a) an EGFRvIII-binding region; (b) a transmembrane region; and (c) a spacer region connecting the trans-membrane region with the EGFRvIII binding region, wherein the spacer region comprises a stalk region comprising at least one dimerization site, and a stalk extension region comprising a sequence with at least 75% sequence identity to the stalk region.

[0071] Further disclosed herein is an engineered effector cell that comprises: (a) an EGFRvIII binding moiety at a surface of said engineered effector cell effective to bind an EGFRvIII molecule or variant thereof, (b) an engineered gene switch polypeptide, wherein the engineered gene switch polypeptide comprises one or more of a transactivation domain, a DNA binding domain, and a ligand binding domain; and (c) a heterologous gene encoding a cytokine, the heterologous gene controlled by a promoter modulated by the engineered gene switch polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0073] FIG. 1 is a schematic depiction of the Sleeping Beauty (SB) system adapted to genetically modify T cells. DNA plasmids expressing a SB transposon system, i.e. SB11, membrane bound IL-15 (mbIL-15), and chimeric antigen receptor (CAR), are transfected to peripheral blood mononuclear cells (PBMC) to redirect T cell specificity. T cells stably expressing integrants on designer activating and propagating cells (AaPC) are propagated and expanded.

[0074] FIG. 2A-2D schematically illustrate various structural components of diverse ligand inducible gene switch vector systems. Exemplary CAR as depicted can include CD19 CAR. EF1A promoter is an exemplary constitutive promoter.

[0075] FIG. 3 is a schematic illustration of a multiple vector system for ligand-inducible gene switch polypeptides.

[0076] FIG. 4 depicts quantitative flow cytometric analysis of cells transfected with ligand inducible gene switch vector systems described herein, on day 1 of post nucleofection in presence/absence of veledimex ligand (solvent: DMSO). Constructs 1, 4, 5, 6, 7 and 9 correspond to constructs as schematically depicted in FIG. 2A-2D.

[0077] FIG. 5 depicts quantitative flow cytometric analysis of cells transfected with ligand inducible gene switch vector systems described herein, on day 21 of post nucleofection in presence/absence of veledimex ligand (solvent: DMSO), gated on CD19-specific CAR positive populations. Ligand was added 48 hours prior to flow cytometric analysis. Constructs 1, 4, 5, 6 and 9 correspond to constructs as schematically depicted in FIG. 2A-2D.

[0078] FIG. 6 depicts quantitative flow cytometric analysis of cells transfected with ligand inducible gene switch vector systems described herein, on day 29 of post nucleofection in presence/absence of veledimex ligand (solvent: DMSO), gated on CD19-specific CAR positive populations. The expression of mbIL-15 is turned on post ligand addition. Constructs 1, 4, 5, 6, 7 and 9 correspond to constructs as schematically depicted in FIG. 2A-2D.

[0079] FIG. 7 shows quantitative flow cytometric analysis of cells transfected with ligand inducible gene switch vector systems described herein, on day 35 of post nucleofection in presence/absence of veledimex ligand (solvent: DMSO), gated on CD19-specific CAR positive populations. The expression of mbIL-15 is turned off post ligand withdrawal. Constructs 1, 4, 5, 6 and 9 correspond to constructs as schematically depicted in FIG. 2A-2D.

[0080] FIG. 8 shows quantitative flow cytometric analysis of cells transfected with ligand inducible gene switch vector systems described herein, on day 40 of post nucleofection in presence/absence of veledimex ligand (solvent: DMSO), gated on CD19-specific CAR positive populations. The expression of mbIL-15 is maintained after re-introduction of ligand. Constructs 1, 4, 5, 6 and 9 correspond to constructs as schematically depicted in FIG. 2A-2D.

[0081] FIG. 9 shows quantitative flow cytometric analysis of cells transfected with ligand inducible gene switch vector systems described herein, on day 48 of post nucleofection in presence/absence of veledimex ligand (solvent: DMSO), gated on CD19-specific CAR positive populations. The expression of mbIL-15 is continuously maintained after re-introduction of ligand. Constructs 1, 4, 5, 6 and 9 correspond to constructs as schematically depicted in FIG. 2A-2D.

[0082] FIG. 10 shows quantitative flow cytometric analysis of cells transfected with ligand inducible gene switch vector systems described herein, on day 50 of post nucleofection in presence/absence of veledimex ligand (solvent: DMSO), gated on CD19-specific CAR positive populations. The expression of mbIL-15 is continuously maintained after re-introduction of ligand. Constructs 1, 4, 5, 6 and 9 correspond to constructs as schematically depicted in FIG. 2A-2D.

[0083] FIG. 11 shows quantitative RT-qPCR analysis of mbIL-15 expression in cells transfected with ligand-inducible gene switch vector systems described herein, on days 29, 33, 35, and 40 of post nucleofection in the presence and absence of a ligand described herein, demonstrating the ligand dependent regulation of expression of mbIL-15 by the gene switch.

[0084] FIG. 12 shows results from a cell survival assay illustrating ligand regulated mbIL-15 expression promotes preferential CAR-T cell survival in the absence of IL-2 and IL-21 cytokines.

[0085] FIG. 13A, 13B, 13C and 13D show the quantitative flow cytometric analysis of cells transfected with ligand-inducible gene switch vector systems described herein, demonstrating veledimex ligand dose response. FIG. 13D shows quantitative flow cytometric analyses of cells transfected with ligand-inducible gene switch vector systems described herein, demonstrating veledimex ligand dose response. The cells were gated on FSC/SSC/LIVE/CD3+. Samples were taken following a 2 day incubation with veledimex at 100nM, 25nM, 6.25nM, 1.56nM, 0.4nM and OnM (DMSO control).

[0086] FIG. 14 is a western blot analysis of mb-IL-15 expression in cells transfected with ligand-inducible gene switch vector systems described herein, in the presence/absence of veledimex ligand.

[0087] FIG. 15A and FIG. 15B are schematic depictions of varying structural components of generic diverse ligand-inducible gene switch vector systems described herein. In the schematic, "PR" stands for a promoter; "IP" is a gene switch ligand-inducible promoter for gene transcription; "5'UTR"is a 5' untranslated region (for transcription into mRNA); "GOI", "GOI 1", "GOI-2" are Gene(s) Of Interest (or first (GOI-1) and second GOI (GOI-2) to be transcribed and expressed as mRNA only (e.g., siRNA) or as mRNA and polypeptide; "Linker" is a cleavable or ribosome skipping linker sequence; "TAD" is a transcription transactivation domain

(for instance, but not limited to a VP16 TAD from Herpes Virus); "LBD" is a nuclear receptor ligand binding domain (for instance, but not limited to a USP domain, an RXR domain, or chimeras (e.g., USP/RXR chimeras) and substitution mutated LBD domains derived from vertebrate and invertebrate species); "DBD" is a DNA binding domain (for instance, but not limited to a Gal4 DBD); "EcR" is an Ecdysone Receptor ligand binding domain, including truncated and substitution mutated EcR domains (such as EcR domains which confer in vitro and in vivo ability to form an active transcriptional activation complex in the presence of steroidal and/or non-steroidal ligands (agonists); including steroidal ligands (such as ponasterone A and muristerone A) and non-steroidal ligands (such as diacyhydrazines, tebufenozide and methoxyfenozide); "Optional GOI"is any optional gene of interest to be transcribed and expressed as mRNA or polypeptide; and "BD polyA" is a Bi-directional Poly-Adenosine tail sequence.

[0088] FIG. 16A and FIG. 16B are schematic depictions of varying structural components of diverse ligand-inducible gene switch vector systems described herein.

[0089] FIG. 17 is a schematic description of an exemplary ligand-inducible gene switch vector system with recombinase attachment sites. Such systems can utilize recombinases (such as seine recombinases) to integrate a single vector into an immune cell (e.g. T cell).

[0090] FIG. 18A is a table depicting % CAR positive T cells over Days 1, 8 and 15. Such T cells were modified to express CAR and gene switch controlled mbIL-15 (RTS-mbIL15) from a single vector using serine recombinase (SF370)-mediated integration. FIG. 18B depicts the induction of mbIl-15 expression in the presence and absence of veledimex at day 15. The figure also demonstrates the loss of mbIL15 expression upon removal of veledimex at day 22. FIG. 18C is an exemplary depiction of RTS-mbIL15- constitutive CAR construct.

[0091] FIG. 19A-B schematically illustrate various structural components of diverse ligand inducible gene switch vector systems under the control of constitutive or T cell specific promoters.

[0092] FIG. 20A schematically illustrates various structural components of diverse ligand inducible gene switch vector systems under the control of constitutive or T cell specific promoters. FIG. 20B-20E show the quantitative flow cytometric analysis of cells transfected with ligand-inducible gene switch vector systems described herein, demonstrating that expression of mbIL-15 is dependent on T cell activation (with addition of ConA) and presence of veledimex. Constructs 1-6 correspond to constructs as schematically depicted in FIG. 20A.

[0093] FIG. 21A-21B depicts various configurations of IL-12.

[0094] FIG. 22 schematically illustrates various structural components of diverse ligand inducible gene switch vector systems under the control of constitutive or inducible promoters.

[0095] FIG. 23 schematically illustrates various structural components of diverse ligand inducible gene switch vector systems to express different forms of IL-12 under the control of constitutive, inducible promoters or tissue-specific promoters (PR). IL-12 can be expressed as a single chain IL-12; membrane bound IL-12 or by individual expression of p35 and p40 domains.

[0096] FIG. 24A-24D schematically illustrate various structural components of diverse ligand inducible gene switch vector systems under the control of constitutive or inducible promoters.

[0097] FIG. 25A-25B schematically illustrate various structural components of diverse ligand inducible gene switch vector systems under the control of constitutive, inducible or T-cell specific promoters.

[0098] FIG. 26 show the quantitative flow cytometric analysis depicting expression of CAR in EGFRvIII CAR T cells expanded ex vivo by successive stimulations via co-culture with AaPC using multiple donors.

[0099] FIG. 27 shows production of IL-12 by cells transfected with ligand-inducible gene switch vector system described herein in presence/absence of veledimex ligand (solvent: DMSO).

[0100] FIG. 28 show analyses of IL-12 expression levels. FIG. 28A shows expression levels of IL-12 by cells transfected with ligand-inducible gene switch vector system described herein in presence/absence of veledimex ligand (solvent: DMSO) in the absence of cell activation. FIG. 28B shows expression levels of IL-12 by cells transfected with ligand-inducible gene switch vector system described herein in presence/absence of veledimex ligand (solvent: DMSO) following antigen specific activation.

[0101] FIG. 29 show analyses of IL-12 expression levels in cells transfected with ligand inducible gene switch vector system described herein and co-cultured in presence/absence of aAPCs and veledimex ligand (solvent: DMSO).

[0102] FIG. 30A and 30B show time course analyses of IL-12 expression levels. FIG. 30 A shows expression levels of IL-12 by cells transfected with ligand-inducible gene switch vector system described herein and co-cultured in absence of AaPC s in presence/absence of veledimex ligand (solvent: DMSO). Ligand was added at 24 hours and withdrawn at 48 hours. FIG. 30B shows expression levels of IL-12 by cells transfected with ligand-inducible gene switch vector system described herein and co-cultured in presence of AaPC s in presence/absence of veledimex ligand (solvent: DMSO). Ligand was added at 24 hours and withdrawn at 48 hours. Expression of of IL-12 was quantified at 24, 48 and 120 hours post addition of ligand.

[0103] FIG. 31A and 31B show analyses of IL-12 expression levels. FIG. 31A shows expression levels of IL-12 in presence/absence of veledimex and AaPCs by cells transfected with ligand-inducible gene switch vector system described herein and expanded by co-culture with

AaPCs in media without veledimex previously(cycle 1). FIG. 31B shows production of IL-12 in presence/absence of veledimex and AaPCs by cells transfected with ligand-inducible gene switch vector system described herein and expanded by co-culture with AaPCs in media previously (cycle 1).

[0104] FIG. 32A and 32B show analyses of IL-12 expression levels. FIG. 32A shows production of IL-12 by cells transfected with ligand-inducible gene switch vector system described herein. Veledimex ligand was added at time 0, withdrawn at time 48 hours, added again at time 168 hours and withdrawn at time 216 hours. FIG. 32B shows expression of CAR in cells transfected with ligand-inducible gene switch vector systems described herein by flow cytometry analysis. Cells are gated on CD3 positive population.

[0105] FIG. 33 shows expression levels of IL-12 by cells transfected with ligand-inducible gene switch vector system described herein in presence of veledimex and in presence (cycle 1)/ absence (cycle 2) of AaPCs.

[0106] FIG. 34 shows veledimex dose dependent expression of IL-12 by cells transfected with ligand-inducible gene switch vector system described herein.

[0107] FIG. 35 shows production of IFNy by CAR-T cells generated via transfection of ligand-inducible gene switch vector system described herein and co-cultured with target cells at 1:1 effector to target cell ratio. Production of IFNy was measured using co-culture of CAR-T cells with EL4, EL4 EGFRvIII target cells as well as no target cell (effector alone) control.

[0108] FIG. 36A and 36B show specific cytotoxicity of CAR-T cells. FIG. 36A shows production of IFNy by CAR-T cells generated via transfection of ligand-inducible gene switch vector system described herein. Effector CAR-T cells were co-cultured with U87MG, U87MG FLuc-GFP, U87MG-EGFRvIII-FLuc-GFP, U251MG, U251MG-FLuc-GFP and U251MG EGFRvIII-FLuc-GFP target cells at 1:1 effector:target cell ratio for 24 hours before analysis of IFNy expression in culture supernatants. FIG. 36B shows % lysis of EGFRvIII expressing glioblastoma cells by CAR-T cells generated via transfection of ligand-inducible gene switch vector system described herein. Effector CAR-T cells were co-incubated with EL4, EL4 expressing EGFRvIII, K562 and K562 expressing EGFRvIII target cell lines at varying effector:target cell ratios.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0109] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope.

[0110] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0111] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0112] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

Definitions

[0113] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0114] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0115] Reference in the specification to "some embodiments," "an embodiment," "one embodiment" or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure.

[0116] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0117] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes 10 and any amounts from 9 to 11. In yet another example, the term "about" in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 30%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0118] The term "isolated" and its grammatical equivalents as used herein refer to the removal of a nucleic acid from its natural environment. The term "purified" and its grammatical equivalents as used herein refer to a molecule or composition, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and/or amplified under laboratory conditions, that has been increased in purity, wherein "purity" is a relative term, not "absolute purity." It is to be understood, however, that nucleic acids and proteins may be formulated with diluents or adjuvants and still for practical purposes be isolated. For example, nucleic acids typically are mixed with an acceptable carrier or diluent when used for introduction into cells. The term "substantially purified" and its grammatical equivalents as used herein refer to a nucleic acid sequence, polypeptide, protein or other compound which is essentially free, i.e., is more than about 50% free of, more than about 70% free of, more than about 90% free of, the polynucleotides, proteins, polypeptides and other molecules that the nucleic acid, polypeptide, protein or other compound is naturally associated with.

[0119] "Polynucleotide" or "oligonucleotide" as used herein refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, triplex DNA, as well as double and single stranded RNA. It also includes modified, for example, by methylation and/or by capping, and unmodified forms of the polynucleotide. The term is also meant to include molecules that include non-naturally occurring or synthetic nucleotides as well as nucleotide analogs.

[0120] "Polypeptide", "peptide", "protein" and their grammatical equivalents as used herein refers to a polymer of amino acid residues. A "mature protein" is a protein which is full-length and which, optionally, includes glycosylation or other modifications typical for the protein in a given cellular environment. Polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4 aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine,3 phenylserine 3-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N' dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2 norbornane)-carboxylic acid, a,y-diaminobutyric acid, a,p-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine. The present disclosure further contemplates that expression of polypeptides described herein in an engineered cell can be associated with post translational modifications of one or more amino acids of the polypeptide constructs. Non limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitylation, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination.

[0121] Nucleic acids and/or nucleic acid sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and/or protein sequences are homologous when their encoding DNAs are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. The homologous molecules can be termed homologs. For example, any naturally occurring proteins, as described herein, can be modified by any available mutagenesis method. When expressed, this mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of identity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology. Methods for determining sequence identity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available.

[0122] The terms "identical" and its grammatical equivalents as used herein or "sequence identity" in the context of two nucleic acid sequences or amino acid sequences of polypeptides refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A "comparison window", as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Apple. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci U.S.A., 85:2444 (1988); by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., ComputerApplications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in MolecularBiology, 24:307-331 (1994). Alignmentisalso often performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 9 8 % 99% or 100% identical to a reference polypeptide, or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 8 5% , 9 0% , 9 8 %, 9 9 % or 100% identical to a reference nucleic acid or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, said percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned.

[0123] The term "substantially identical" and its grammatical equivalents as applied to nucleic acid or amino acid sequences mean that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, at least 95%, at least 98% and at least 99%, compared to a reference sequence using the programs described above, e.g., BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. In embodiments, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, over a region of at least about 100 residues, and in embodiments, the sequences are substantially identical over at least about 150 residues. In embodiments, the sequences are substantially identical over the entire length of the coding regions.

[0124] "Transposon" or "transposable element" (TE) is a vector DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and altering the cell's genome size. Transposition often results in duplication of the TE. Class I TEs are copied in two stages: first, they are transcribed from DNA to RNA, and the RNA produced is then reverse transcribed to DNA. This copied DNA is then inserted at a new position into the genome. The reverse transcription step is catalyzed by a reverse transcriptase, which may be encoded by the TE itself. The characteristics of retrotransposons are similar to retroviruses, such as HIV. The cut-and-paste transposition mechanism of class II TEs does not involve an RNA intermediate. The transpositions are catalyzed by several transposase enzymes. Some transposases non-specifically bind to any target site in DNA, whereas others bind to specific DNA sequence targets. The transposase makes a staggered cut at the target site resulting in single-strand 5' or 3' DNA overhangs (sticky ends). This step cuts out the DNA transposon, which is then ligated into a new target site; this process involves activity of a DNA polymerase that fills in gaps and of a DNA ligase that closes the sugar-phosphate backbone. This results in duplication of the target site. The insertion sites of DNA transposons may be identified by short direct repeats which may be created by the staggered cut in the target DNA and filling in by DNA polymerase, followed by a series of inverted repeats important for the TE excision by transposase. Cut-and-paste TEs may be duplicated if their transposition takes place during S phase of the cell cycle when a donor site has already been replicated, but a target site has not yet been replicated. Transposition can be classified as either "autonomous" or "non-autonomous" in both Class I and Class II TEs. Autonomous TEs can move by themselves while non-autonomous TEs require the presence of another TE to move. This is often because non-autonomous TEs lack transposase (for class II) or reverse transcriptase (for class I).

[0125] "Transposase" refers an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. In some embodiments, the transposase's catalytic activity can be utilized to move gene(s) from a vector to the genome. In some embodiments, the transposase's catalytic activity can be utilized to move gene(s) from a vector (e.g. a transposon) to the genome. In certain embodiments, the Sleeping Beauty transposase is provided as an mRNA. In some aspects, the mRNA comprises a cap and a poly-A tail.

[0126] The nucleic acid sequences and vectors disclosed or contemplated herein may be introduced into a cell by "transfection," "transformation," "nucleofection" or "transduction." "Transfection," "transformation," or "transduction," as used herein refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)); and nucleofection (Trompeter et al., J. Immunol. Methods 274:245-256 (2003). Phage or viral vectors can be introduced into host cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available.

[0127] "Tumor antigen" as used herein refers to any antigenic substance produced or overexpressed in tumor cells. It may, for example, trigger an immune response in the host. Alternatively, for purposes of this disclosure, tumor antigens may be proteins that are expressed by both healthy and tumor cells but because they identify a certain tumor type, are a suitable therapeutic target. In embodiments, the tumor antigen is CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein,

GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 or VEGF-R2. In one embodiment, the tumor antigen is EGFRvIII, which is a target for CAR T cell therapies for treating myeloid malignancies, for example, glioblastoma or glioblastoma multiforme (GBM). In another embodiment, the tumor antigen is CD19. In yet another embodiment, the tumor antigen is CD33.

[0128] The term "enhancer," as used herein, refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences. The term "Ig enhancers" refers to enhancer elements derived from enhancer regions mapped within the immunoglobulin (Ig) locus (such enhancers include for example, the heavy chain (mu) 5' enhancers, light chain (kappa) 5' enhancers, kappa and mu intronic enhancers, and 3' enhancers (see generally Paul W. E. (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363; and U.S. Pat. No. 5,885,827).

[0129] "Coding sequence" as used herein refers to a segment of a polynucleotide that codes for a polypeptide. The region or sequence is bounded nearer the 5' end by a start codon and nearer the 3' end with a stop codon. Coding sequences may also be referred to as open reading frames.

[0130] "Operably linked" as used herein refers to refers to the physical and/or functional linkage of a DNA segment to another DNA segment in such a way as to allow the segments to function in their intended manners. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is linked to the regulatory sequence, such as, for example, promoters, enhancers and/or silencers, in a manner which allows modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter, in the correct reading frame with respect to the transcription initiation site and allows transcription elongation to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence coding for a gene product when it is ligated to the DNA sequence in such a manner as to increase or decrease, respectively, the transcription of the DNA sequence. Enhancers and silencers may be located upstream, downstream or embedded within the coding regions of the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for a polypeptide if the signal sequence is expressed as a preprotein that participates in the secretion of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or via adapters or linkers inserted in the sequence using restriction endonucleases known to one of skill in the art.

[0131] The term "transcriptional regulator" refers to a biochemical element that acts to prevent or inhibit the transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or to permit or stimulate the transcription of the promoter-driven DNA sequence under certain environmental conditions (e.g., an inducer or an enhancer).

[0132] The term "induce", "induction" and its grammatical equivalents as used herein refer to an increase in nucleic acid sequence transcription, promoter activity and/or expression brought about by a transcriptional regulator, relative to some basal level of transcription.

[0133] A "target" gene or "heterologous" gene, or "gene of interest (GOI)" refers to a gene introduced into the host cell by gene transfer. Exemplary GOI can be an antigen binding polypeptide that can include an antigen binding polypeptide, chimeric receptor, CAR,TCR, a cytokine and/or a cell tag as described herein.

[0134] "Recombinase" as used herein refers to a group of enzymes that can facilitate site specific recombination between defined sites, where the sites are physically separated on a single DNA molecule or where the sites reside on separate DNA molecules. The DNA sequences of the defined recombination sites are not necessarily identical. Initiation of recombination depends on protein-DNA interaction, within the group there are large number of proteins that catalyze phage integration and excision (e.g., X integrase, .4C31), resolution of circular plasmids (e.g., Tn3, gamma delta, Cre, Flp), DNA inversion for expression of alternate genes (e.g., Hin, Gin, Pin), assembly of genes during development (e.g., Anabaena nitrogen fixation genes), and transposition (e.g., IS607 transposon). Most site-specific recombinases fall into one of the two families, based on evolutionary and mechanistic relatedness. These are . X integrase family or tyrosine recombinases (e.g., Cre, Flp, Xer D) and resolvase/integrase family or serine recombinase family (e.g., §C31, TP901-1, Tn3, gamma delta).

[0135] "Recombination attachment sites" are specific polynucleotide sequences that are recognized by the recombinase enzymes described herein. Typically, two different sites are involved (termed "complementary sites"), one present in the target nucleic acid (e.g., a chromosome or episome of a eukaryote or prokaryote) and another on the nucleic acid that is to be integrated at the target recombination site. The terms "attB" and "attP," which refer to attachment (or recombination) sites originally from a bacterial target and a phage donor, respectively, are used herein although recombination sites for particular enzymes may have different names. The recombination sites typically include left and right arms separated by a core or spacer region. Thus, an attB recombination site consists of BOB', where B and B' are the left and right arms, respectively, and 0 is the core region. Similarly, attP is POP', where P and P' are the arms and 0 is again the core region. Upon recombination between the attB and attP sites, and concomitant integration of a nucleic acid at the target, the recombination sites that flank the integrated DNA are referred to as "attL" and "attR." The attL and attR sites, using the terminology above, thus consist of BOP' and POB', respectively. In some representations herein, the "0"is omitted and attB and attP, for example, are designated as BB' and PP', respectively.

Modulation of the Expression of Genes

[0136] In the field of genetic engineering, precise control of gene expression is a valuable tool for studying, manipulating, and controlling development and other physiological processes. Gene expression is a complex biological process involving a number of specific protein-protein interactions. Tightly regulated inducible gene expression systems or "gene switches" are useful for various applications such as gene therapy, large scale production of proteins in cells, cell based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals. However, for therapy, regulated and localized expression is important to prevent off-target effects. Provided herein are means to regulate the expression of heterologous genes such as cytokines when required and at a specific vicinity of a target cell or location. Further provided herein is a method to regulate the expression of heterologous genes such as cytokines wherein the cytokine, for example, is under the control of a ligand inducible promoter. In some instances, the method results in a low or no basal level of heterologous gene expression in the absence of the ligand. In a further embodiment, the ligand inducible promoter is a gene switch ligand inducible promoter. Further provided herein are methods to regulate the expression of heterologous genes such as cytokines from an engineered cell. Herein an engineered cell is a cell which has been modified from its natural or endogenous state. An example of an engineered cell is a cell described herein which has been modified (e.g., by transfection of a polynucleotide into the cell). In some instances, the engineered cell can be an engineered immune effector cell. In one instance, the engineered cell is a T cell. In another instance, the engineered cell is a NK cell. In one instance, engineered cells are activated prior to inducing the expression of the heterologous gene with a ligand. In another instance, the engineered cells are activated by exposing the cells to an antigen. The antigen can be an antigen that is recognized by the antigen binding polypeptide expressed by the engineered cell. The antigen can be a tumor antigen or an infectious disease antigen. In one instance, the engineered cell can comprise an antigen binding polypeptide that binds such an antigen.

[0137] In a further instance, the gene switch components of a gene switch ligand inducible promoter can be further regulated by a tissue specific promoter. In such cases, the gene switch components are only expressed when the tissue specific promoter is activated, for example, in an activated T cell or an activated NK cell.

Vector

[0138] An "expression vector" or "vector" is any genetic element, e.g., a plasmid, chromosome, virus, transposon, behaving either as an autonomous unit of polynucleotide replication within a cell. (i.e. capable of replication under its own control) or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages and cosmids. Vectors may contain polynucleotide sequences which are necessary to effect ligation or insertion of the vector into a desired host cell and to effect the expression of the attached segment. Such sequences differ depending on the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences and transcription and translation termination sequences. Alternatively, expression vectors may be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences.

[0139] Vector also can comprise a "selectable marker gene." The term "selectable marker gene," as used herein, refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected for or against, in the presence of a corresponding selective agent. Suitable selectable marker genes are known in the art and described in, e.g., International Patent Application Publications WO 1992/08796 and WO 1994/28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981); Santerre et al., Gene, 30: 147 (1984); Kent et al., Science, 237: 901-903 (1987); Wigler et al., Cell, 11: 223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026 (1962); Lowy et al., Cell, 22: 817 (1980); and U.S. Pat. Nos. 5,122,464 and 5,770,359.

[0140] In some embodiments, the vector is an "episomal expression vector" or "episome," which is able to replicate in a host cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Barr

Nuclear Antigen 1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK CMV from Stratagene (La Jolla, Calif) represent non-limiting examples of an episomal vector that uses T-antigen and the SV40 origin of replication in lieu of EBNA1 and oriP.

[0141] As used herein, "a gene expression cassette" is part of a vector and can contain constitutive promoters such as EF la or inducible promoters, 5' UTR, 3' UTR and polyA elements and one or more genes of interest (GOI). In one embodiment, the polyA element is SV40e polyA (SEQ ID NO 65). In another embodiment, the polyA element is bidirectional aCA polyA (SEQ ID NO 66). In yet another embodiment, the polyA is PA2 polyA (SEQ ID NO 67). In some aspects, a gene expression cassette may further comprise gene switch components such as a DNA-binding domain fused to a nuclear receptor ligand binding domain and/or a transactivation domain fused to a nuclear receptor ligand binding domain. A vector can include one or more gene expression cassette(s).

Vector Modifications

[0142] A polynucleotide vector useful for the methods and compositions described herein can be a good manufacturing practices (GMP) compatible vector. For example, a GMP vector may be purer than a non-GMP vector. In some cases, purity can be measured by bioburden. For example, bioburden can be the presence or absence of aerobes, anaerobes, sporeformers, fungi, or combinations thereof in a vector composition. In some cases, a pure vector can be endotoxin low or endotoxin free. Purity can also be measured by double-stranded primer-walking sequencing. Plasmid identity can be a source of determining purity of a vector. A GMP vector of the present disclosure can be from 10% to 99% more pure than a non-GMP vector. A GMP vector can be from 10%, 15%,20%,25%,30%,35%, 40%,45%, 50%,55%, 60%,65%,70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% more pure than a non-GMP vector as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof.

[0143] In some cases, a terminator sequence at the end of the first gene program is used. A terminator sequence can ensure that a transcript is terminating prior to initiating a second gene program. For example, an expression vectors may contain sequences necessary for the termination of transcription and for stabilizing an mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions can contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA. Cells comprising the expression vector are grown under conditions that provide for expression of the desired polypeptide, either in vivo or in vitro.

[0144] In some cases, a spacer sequence can be used at the end of a first polypeptide encoded by a polynucleotide in a vector. In other cases, a spacer sequence can be used at the end of a second gene in a vector. A spacer sequence can also be used following a first gene and a second gene in a vector.

[0145] These vectors can be used to express a polypeptide encoded by a gene, or portion of a gene of interest. A gene of portion or a gene can be inserted by using any method, viral or non viral. For example; a method can be a non-viral based technique.

IRES Elements

[0146] Also disclosed herein are constructs that comprise an IRES element to facilitate the expression and functionality of the polynucleotides and polypeptides described herein. The term "internal ribosome entry site (RES)" as used herein can be intended to mean internal ribosomal entry site. In a vector comprising an RES sequence, a first gene can be translated by a cap dependent, ribosome scanning, mechanism with its own 5'-UTR, whereas translation of a subsequent gene can be accomplished by direct recruitment of a ribosome to an IRES in a cap independent manner. An RES sequence can allow eukaryotic ribosomes to bind and begin translation without binding to a 5'capped end. An IRES sequence can allow expression of multiple genes from one transcript (Mountford and Smith 1995).

[0147] The term "CAP" or "cap" as used herein refers to a modified nucleotide, generally a 7 methyl guanosine, linked 3'to 5'(7meG-ppp-G), to the 5'end of a eukaryotic mRNA, that serves as a required element in the normal translation initiation pathway during expression of protein from that mRNA.

[0148] In certain cases, an IRES region can be derived from a virus, such as picornavirus, encephalomyocarditis virus, hepatitis C virus IRES sequence. In other cases, an RES sequence can be derived from an encephalomyocarditis virus. The term "EMCV" or "encephalomyocarditis virus" as used herein refers to any member isolate or strain of the encephalomyocarditis virus species of the genus of the family Picornaviridae. Examples are: EMCV-R (Rueckert) strain virus, Columbia-SK virus. In some cases, a cellular IRES element, such as eukaryotic initiation factor 4G, immunoglobulin heavy chain binding protein, c-myc proto-oncogene, vascular endothelial growth factor, fibroblast growth factor-i IRES, or any combination or modification thereof can be used. In some cases, a cellular IRES can have increased gene expression when compared to a viral IRES.

[0149] An IRES sequence of viral, cellular or a combination thereof can be utilized in a vector. An RES can be from encephalomyocarditis (EMCV) or poliovirus (PV). In some cases, an IRES element is selected from a group consisting of Poliovirus (PV), Encephalomyelitis virus

(EMCV), Foot-and-mouth disease virus (FMDV), Porcine teschovirus-i (PTV-1), Aichivirus (AiV), Seneca Valley virus (SVV), Hepatitis C virus (HCV), Classical swine fever virus (CSFV), Human immunodeficiency virus-2 (HIV-2), Human immunodeficiency virus-i (HIV-1), Moloney murine leukemia virus (MoMLV), Feline immunodeficiency virus (FIV), Mouse mammary tumor virus (MMTV), Human cytomegalovirus latency (pUL138), Epstein-Barr virus (EBNA-1), Herpes virus Marek's disease (MDV RLORF9), SV40 polycistronic 19S (SV40 19S), Rhopalosiphum padi virus (RhPV), Cricket paralysis virus (CrPV), Ectropis obliqua picorna-like virus (EoPV), Plautia stali intestine virus (PSIV), Triatoma virus (TrV), Bee paralysis dicistrovirus (IAPV, KBV), Black currant reversion virus (BRV), Pelargonium flower break virus (PFBV), Hibiscus chlorotic ringspot virus (HCRSV), Crucifer-infecting tobamovirus (CrTMV), Potato leaf roll polerovirus (PLRV), Tobacco etch virus (TEV), Giardiavirus (GLV), Leishmania RNA virus-i (LRV-1), and combinations or modifications thereof In some cases, an IRES is selected from a group consisting of Apaf-1, XIAP, HIAP2/c-IAP1, DAP5, Bcl-2, c-myc, CAT-1, INR, Differentiation LEF-1, PDGF2, HIF-i a, VEGF, FGF2, BiP, BAG-1, CIRP, p53, SHMT1, PITSLREp58, CDK, Rpr, hid, hsp70, grim, skl, Antennapedia, dFoxO, dInR, Adh Adhr, HSPiOi, ADH, URE-2,GPR, NCE102, YMR181a, MSNi, BOI, FLO8, GICi, and any combination or modification thereof When an IRES element is included between two open reading frames (ORFs), initiation of translation can occur by a canonical 5'- m7GpppN cap dependent mechanism in a first ORF and a cap-independent mechanism in a second ORF downstream of the IRES element.

[0150] In some cases, genes can be linked by an internal ribosomal entry site (IRES). An IRES can allow simultaneous expression of multiple genes. For example, an IRES sequence can permit production of multiple proteins from a single mRNA transcript. A ribosome can bind to an IRES in a 5'-cap independent manner and initiate translation.

[0151] In some cases, an IRES sequence can be or can be about 500 base pairs. An IRES sequence can be from 300 base pairs to 1000 base pairs. For example, an IRES can be 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 base pairs long.

[0152] In some cases, expression of a downstream gene within a vector comprising an IRES sequence can be reduced. For example, a gene following an IRES sequence can have reduced expression over a gene preceding an IRES sequence. Reduced expression can be from 1% to 99% reduction over a preceding gene.

[0153] In certain embodiments, an IRES is an EMCV IRES comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 9 9 .5% or 100% identity with the nucleotide sequence of SEQ ID NO 18.

Linkers

[0154] Also disclosed are constructs that comprise a linker to facilitate the expression and functionality of the polynucleotides and polypeptides described herein. In some embodiments, a polynucleotide linker can be utilized in a polynucleotide described herein. A polynucleotide linker can be a double-stranded segment of DNA containing desired restriction sites that may be added to create end structures that are compatible with a vector comprising a polynucleotide described herein. In some cases, a polynucleotide linker can be useful for modifying vectors comprising polynucleotides described herein. For example, a vector modification comprising a polynucleotide linker can be a change in a multiple cloning site, or the addition of a poly histidine tail. Polynucleotide linkers can also be used to adapt the ends of blunt insert DNA for cloning into a vector cleaved with a restriction enzyme with cohesive end termini. The use of polynucleotide linkers can be more efficient than a blunt ligation into a vector and can provide a method of releasing an insert from a vector in downstream applications. In some cases an insert can be a polynucleotide sequence encoding polypeptides useful for therapeutic applications. In some cases, a linker can be a cleavable linker.

[0155] A polynucleotide linker can be an oligomer. A polynucleotide linker can be a DNA double strand, single strand, or a combination thereof. In some cases, a linker can be RNA. A polynucleotide linker can be ligated into a vector comprising a polynucleotide described herein by a T4 ligase in some cases. To facilitate a ligation an excess of polynucleotide linkers can be added to a composition comprising an insert and a vector. In some cases, an insert and vector are pre-treated before a linker is introduced. For example, pre-treatment with a methylase can prevent unwanted cleavage of insert DNA.

[0156] In certain embodiments, two or more polypeptides encoded by a polynucleotide described herein can be separated by an intervening sequence encoding an intervening linker polypeptide. Herein the term "intervening linker polypeptide" referring to an amino acid sequence separating two or more polypeptides encoded by a polynucleotide is distinguished from the term "peptide linker" which refers to the sequence of amino acids which is optionally included in a polypeptide construct disclosed herein to connect the transmembrane domain to the cell surface polypeptide (e.g., comprising a truncated variant of a natural polypeptide). In certain cases, the intervening linker is a cleavage-susceptible intervening linker polypeptide. In some embodiments, the linker is a cleavable or ribosome skipping linker. In some embodiments, the cleavable linker or ribosome skipping linker sequence is selected from the group consisting of 2A, GSG-2A, GSG linker, SGSG linker, furinlink variants and derivatives thereof. In some embodiments, the 2A linker is a p2A linker, a T2A linker, F2A linker or E2A linker. In some embodiments, polypeptides of interest are expressed as fusion proteins linked by a cleavage susceptible intervening linker polypeptide. In certain embodiments, cleavage-susceptible intervening linker polypeptide(s) can be any one or more of F/T2A, T2A, p2A, 2A, GSG-p2A, GSG linker, and furinlink variants. In certain embodiments, the linker polypeptide comprises disclosed in the table below:

Table 1. Linker amino acid sequences and polynucleotide sequences SEQ SEQ Polynucleotide Sequence (5' to Amino Acids Sequence (5' to Linker Name ID ID NO 3' where applicable) NO 3' where applicable) GGCAGCACCTCCGGCAGCG Whitlow Linker 1 GCAAGCCTGGCAGCGGCGA 146 GSTSGSGKPGSGEGSTKG GGGCAGCACCAAGGGC TCTGGCGGAGGATCTGGAG GAGGCGGATCTGGAGGAGG SGGGSGGGGSGGGGSGGG Linker 2 AGGCAGTGGAGGCGGAGGA 147 GSGGGSLQ TCTGGCGGAGGATCTCTGC AG GSG linker 3 GGAAGCGGA 148 GSG SGSG linker 4 AGTGGCAGCGGC 149 SGSG GGTGGCGGTGGCTCGGGCG (G4S)3 linker 5 GTGGTGGGTCGGGTGGCGG 150 GGGGSGGGGSGGGGS CGGATCT

ie Furin lavage 6 CGTGCAAAGCGT 151 RAKR AGAGCCAAGAGGGCACCGG TGAAACAGACTTTGAATTTT RAKRAPVKQTLNFDLLKL Fmdv 7 GACCTTCTGAAGTTGGCAG 152 AGDVESNPGP GAGACGTTGAGTCCAACCC TGGGCCC Thosea asigna GAGGGCAGAGGAAGTCTGC virus 2A region 8 TAACATGCGGTGACGTCGA 153 EGRGSLLTCGDVEENPGP (T2A) GGAGAATCCTGGACCT AGAGCTAAGAGGGGAAGCG Furin-GSG-T2A 9 GAGAGGGCAGAGGAAGTCT 154 RAKRGSGEGRGSLLTCGD GCTAACATGCGGTGACGTC VEENPGP GAGGAGAATCCTGGACCT AGGGCCAAGAGGAGTGGCA GCGGCGAGGGCAGAGGAA RAKRSGSGEGRGSLLTCGD Furin-SGSG-T2A 10 GTCTTCTAACATGCGGTGAC 155 VEENPGP GTGGAGGAGAATCCCGGCC CT Porcine GCAACGAACTTCTCTCTCCT teschovirus-1 2A 11 AAAACAGGCTGGTGATGTG 156 ATNFSLLKQAGDVEENPGP region (P2A) GAGGAGAATCCTGGTCCA GGAAGCGGAGCTACTAACT GSGATNFSLLKQAGDVEE GSG-P2A 12 TCAGCCTGCTGAAGCAGGC 157 NPGP TGGAGACGTGGAGGAGAAC

SEQ SEQ Polynucleotide Sequence (5' to Amino Acids Sequence (5' to Linker Name ID ID NO 3' where applicable) NO 3' where applicable) CCTGGACCT Equine rhinitisA CAGTGTACTAATTATGCTCT virus2A region 13 CTTGAAATTGGCTGGAGAT 158 QCTNYALLKLAGDVESNP vu2A rGTTGAGAGCAACCCTGGAC GP (E2A)CT

Foot-and-mouth GTCAAACAGACCCTAAACT diseasevirus2A 14 TTGATCTGCTAAAACTGGCC 159 VKQTLNFDLLKLAGDVES deviru2A 1 GGGGATGTGGAAAGTAATC NPGP region (F2A) CCGGCCCC CGTGCAAAGCGTGCACCGG TGAAACAGGGAAGCGGAGC RAKRAPVKQGSGATNFSLL FP2A 15 TACTAACTTCAGCCTGCTGA 160 KQAGDVEENPGP AGCAGGCTGGAGACGTGGA GGAGAACCCTGGACCT Linker-GSG 16 GCACCGGTGAAACAGGGAA 161 APVKQGSG GCGGA Linker 17 GCACCGGTGAAACAG 162 APVKQ

[0157] In some embodiments, an intervening linker polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or 100% identity with the amino acid sequence of Whitlow linker (SEQ ID NO 146), GSG linker (SEQ ID NO 148), SGSG linker (SEQ ID NO 149), (G4S)3 linker (SEQ ID NO 150), Furin cleavage site / FurlinkI (SEQ ID NO 151), Fmdv linker (SEQ ID NO 152), Thosea asigna virus 2A region (T2A) (SEQ ID NO 153), Furin-GSG-T2A (SEQ ID NO 154), Furin-SGSG-T2A (SEQ ID NO 155), porcine teschovirus-1 2A region (P2A) (SEQ ID NO 156), GSG-P2A (SEQ ID NO 157), equine rhinitis A virus 2A region (E2A) (SEQ ID NO 158), or foot-and-mouth disease virus 2A region (F2A) (SEQ ID NO: 159). In some cases, an intervening linker polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 9 9 %, 99.5% or 100% identity with the amino acid sequence of linkers (SEQ ID NOS 147, l6lor 162) In some cases, a viral 2A sequence can be used. 2A elements can be shorter than RES, having from 5 to 100 base pairs. In some cases, a 2A sequence can have 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 nucleotides in length. 2A linked genes can be expressed in one single open reading frame and "self-cleavage" can occur co-translationally between the last two amino acids, GP, at the C-terminus of the 2A polypeptide, giving rise to equal amounts of co-expressed proteins.

[0158] A viral 2A sequence can be about 20 amino acids. In some cases, a viral 2A sequence can contain a consensus motif Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro. A consensus motif sequence can act co-translationally. For example, formation of a normal peptide bond between a glycine and proline residue can be prevented, which can result in ribosomal skipping and cleavage of a nascent polypeptide. This effect can produce multiple genes at equimolar levels.

[0159] A 2A peptide can allow translation of multiple proteins in a single open reading frame into a polypeptide that can be subsequently cleaved into individual polypeptide through a ribosome-skipping mechanism (Funston, Kallioinen et al. 2008). In some embodiments, a 2A sequence can include: F/T2A, T2A, p2A, 2A, T2A, E2A, F2A, and BmCPV2A, BmIFV2A, and any combination thereof.

[0160] In some cases, a vector can comprise an RES sequence and a 2A linker sequence. In other cases, expression of multiple genes linked with 2A peptides can be facilitated by a spacer sequence (GSG) ahead of the 2A peptides. In some cases, constructs can combine a spacers, linkers, adaptors, promotors, or combinations thereof. For example, a linker can have a spacer (SGSG or GSG or Whitlow linker) and furin linker (R-A-K-R) cleavage site with different 2A peptides. A spacer can be an I-Ceui. In some cases, a linker can be engineered. For example, a linker can be designed to comprise chemical characteristics such as hydrophobicity. In some cases, at least two linker sequences can produce the same protein. In other cases, multiple linkers can be used in a vector. For example, genes of interest can be separated by at least two linkers, as shown in FIG 2 and FIG 3.

[0161] In certain embodiments, two or more polypeptides encoded by a polynucleotide described herein can be separated by an intervening sequence encoding a linker polypeptide. In certain cases, the linker is a cleavage-susceptible linker. In some embodiments, polypeptides of interest are expressed as fusion proteins linked by a cleavage-susceptible linker polypeptide. In certain embodiments, cleavage-susceptible linker polypeptide(s) can be any one or two of: Furinlink, fmdv, p2a, GSG-p2a, and/or fp2a described below. In some cases, a linker is APVKQGSG (SEQ ID NO 161).

[0162] In certain cases, a linker polypeptide can comprise an amino acid sequence "RAKR" (SEQ ID NO 151). In certain cases, a Furin linker polypeptide can be encoded by a polynucleotide sequence polynucleotide sequence comprising "CGTGCAAAGCGT" (SEQ ID NO 6) or "AGAGCTAAGAGG." (SEQ ID NO 9).

[0163] In some embodiments, a linker can be utilized in a polynucleotide described herein. A linker can be a flexible linker, a rigid linker, an in vivo cleavable linker, or any combination thereof. In some cases, a linker may link functional domains together (as in flexible and rigid linkers) or releasing free functional domain in vivo as in in vivo cleavable linkers.

[0164] Linkers may improve biological activity, increase expression yield, and achieving desirable pharmacokinetic profiles. A linker can also comprise hydrazone, peptide, disulfide, or thioesther.

[0165] In some cases, a linker sequence described herein can include a flexible linker. Flexible linkers can be applied when a joined domain requires a certain degree of movement or interaction. Flexible linkers can be composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. A flexible linker can have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linker). An example of a flexible linker can have the sequence of (Gly Gly-Gly-Gly-Ser)n (SEQ ID NO 150). By adjusting the copy number "n", the length of this exemplary GS linker can be optimized to achieve appropriate separation of functional domains, or to maintain necessary inter-domain interactions. Besides GS linkers, other flexible linkers can be utilized for recombinant fusion proteins. In some cases, flexible linkers can also be rich in small or polar amino acids such as Gly and Ser, but can contain additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu can be used to improve solubility.

[0166] Flexible linkers included in linker sequences described herein, can be rich in small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. Flexible linkers can be suitable choices when certain movements or interactions are desired for fusion protein domains. In addition, although flexible linkers may not have rigid structures, they can serve as a passive linker to keep a distance between functional domains. The length of flexible linkers can be adjusted to allow for proper folding or to achieve optimal biological activity of the fusion proteins.

[0167] A linker described herein can further include a rigid linker in some cases. A rigid linker may be utilized to maintain a fixed distance between domains of a polypeptide. Examples of rigid linkers can be: Alpha helix-forming linkers, Pro-rich sequence, (XP)n, X-Pro backbone, A(EAAAK)nA (n = 2-5), to name a few. Rigid linkers can exhibit relatively stiff structures by adopting a-helical structures or by containing multiple Pro residues in some cases.

[0168] A linker described herein can be cleavable in some cases. In other cases a linker is not cleavable. Linkers that are not cleavable may covalently join functional domains together to act as one molecule throughout an in vivo processes or an ex vivo process. A linker can also be cleavable in vivo. A cleavable linker can be introduced to release free functional domains in vivo. A cleavable linker can be cleaved by the presence of reducing reagents, proteases, to name a few. For example, a reduction of a disulfide bond may be utilized to produce a cleavable linker. In the case of a disulfide linker, a cleavage event through disulfide exchange with a thiol, such as glutathione, could produce a cleavage. In other cases, an in vivo cleavage of a linker in a recombinant fusion protein may also be carried out by proteases that can be expressed in vivo under pathological conditions (e.g. cancer or inflammation), in specific cells or tissues, or constrained within certain cellular compartments. In some cases, a cleavable linker may allow for targeted cleavage. For example, the specificity of many proteases can offer slower cleavage of a linker in constrained compartments. A cleavable linker can also comprise hydrazone, peptides, disulfide, or thioesther. For example, a hydrazone can confer serum stability. In other cases, a hydrazone can allow for cleavage in an acidic compartment. An acidic compartment can have a pH up to 7. A linker can also include a thioether. A thioether can be nonreducible A thioether can be designed for intracellular proteolytic degradation.

[0169] In certain embodiments, an fmdv linker polypeptide comprises a sequence that can be at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 9 7 %, 98%, 9 9 % or

100% identical to SEQ ID NO 152. In certain embodiments, an fmdv linker polypeptide is one or more of the linkers encoded in a single vector linking two or more fusion proteins. In certain cases, an fmdv linker polypeptide can be encoded by a polynucleotide open reading frame (ORF) nucleic acid sequence. In some cases, an ORF encoding fmdv comprises or consists of a sequence of SEQ ID NO 7). In certain embodiments, a polynucleotide encoding fmdv is at least 45%,50%,55%, 60%,65%,70%,75%, 80%,85%,90%,95%, 97%,98%,99% or 100% identical to SEQ ID NO 7).

[0170] In certain cases, a linker polypeptide can be a "p2a" linker. In certain embodiments, a p2a polypeptide can comprise a sequence that can be about at least 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%,85%,90%,95%, 97%, 98%,99% or 100% identical to SEQ IDNO 156). In certain embodiments, the p2a linker polypeptide can be one or more of the linkers encoded in a single vector linking two or more fusion proteins. In some cases, a p2a linker polypeptide can be encoded by a polynucleotide open reading frame (ORF) nucleic acid sequence. In certain embodiments, an ORF encoding p2a comprises or consists of the sequence of SEQ ID NO 11). In certain cases, a polynucleotide encoding p2a can be or can be about at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 11).

[0171] In some cases, a linker polypeptide can be a "GSG-p2a" linker. In certain embodiments, a GSG-p2a linker polypeptide can comprise a sequence that can be about at least 45%, 50%,55%, 60%,65%,70%,75%, 80%,85%,90%,95%, 97%,98%,99% or 100% identical to SEQ ID NO 157). In certain embodiments, a GSG-p2a linker polypeptide can be one or more of the linkers encoded in a single vector linking two or more fusion proteins. In some cases, a GSG-p2a linker polypeptide can be encoded by a polynucleotide open-reading frame (ORF) nucleic acid sequence. An ORF encoding GSG p2a can comprise the sequence of SEQ ID

NO 12). In some cases, a polynucleotide encoding GSG-p2a can be or can be about at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 12).

[0172] A linker polypeptide can be an "fp2a" linker as provided herein. In certain embodiments, a fp2a linker polypeptide can comprise a sequence that can be about at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 160). In certain cases, an fp2a linker polypeptide can be one or more of the linkers encoded in a single vector linking two or more fusion proteins. In some cases, a fp2a linker polypeptide can be encoded by a polynucleotide open reading frame (ORF) nucleic acid sequence. In certain embodiments, a polynucleotide encoding an fp2a linker can be or can be about at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 15).

[0173] In some cases, a linker can be engineered. For example, a linker can be designed to comprise chemical characteristics such as hydrophobicity. In some cases, at least two linker sequences can produce the same protein. A sequence can be or can be about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 9 6 %, 97%, 9 8 %, 9 9 %, or 100% identical to a polypeptide sequence of SEQ ID NOS 147, 161 or 162. In other cases, multiple linkers can be used in a vector. For example, genes of interest, and one or more gene switch polypeptide sequences described herein can be separated by at least two linkers, as shown in FIG 15 and FIG 16. In some cases, genes can be separated by 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 linkers.

[0174] A linker can be an engineered linker. Methods of designing linkers can be computational. In some cases, computational methods can include graphic techniques. Computation methods can be used to search for suitable peptides from libraries of three dimensional peptide structures derived from databases. For example, a Brookhaven Protein Data Bank (PDB) can be used to span the distance in space between selected amino acids of a linker.

[0175] In some embodiments are polynucleotides encoding a polypeptide construct comprising a furin polypeptide and a 2A polypeptide, wherein the furin polypeptide and the 2A polypeptide are connected by a polypeptide linker comprising at least three hydrophobic amino acids. In some cases, at least three hydrophobic amino acids are selected from the list consisting of glycine (Gly)(G), alanine (Ala)(A), valine (Val)(V), leucine (Leu)(L), isoleucine (Ile)(I), proline (Pro)(P), phenylalanine (Phe)(F), methionine (Met)(M), tryptophan (Trp)(W). In some cases, a polypeptide linker may also include one or more GS linker sequences, for instance (GS)n, (SG)n, (GSG)n (SEQ ID NO: 78) and (SGSG)n (SEQ ID NO: 79) wherein n can be any number from zero to fifteen.

[0176] Provided are methods of obtaining an improved expression of a polypeptide construct comprising: providing a polynucleotide encoding said polypeptide construct comprising a first functional polypeptide and a second functional polypeptide, wherein said first functional polypeptide and second functional polypeptide are connected by a linker polypeptide comprising a sequence with at least 60% identity to the sequence APVKQ (SEQ ID NO 162); and expressing said polynucleotide in a host cell, wherein said expressing results in an improved expression of the polypeptide construct as compared to a corresponding polypeptide construct that does not have a linker polypeptide comprising a sequence with at least 60% identity to the sequence APVKQ.

Promoters

[0177] "Promoter" refers to a region of a polynucleotide that initiates transcription of a coding sequence. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand). Some promoters are constitutive as they are active in all circumstances in the cell, while others are regulated becoming active in response to specific stimuli, e.g., an inducible promoter. Yet other promoters are tissue specific or activated promoters, including but not limited to T-cell specific promoters.

[0178] The term "promoter activity" and its grammatical equivalents as used herein refer to the extent of expression of nucleotide sequence that is operably linked to the promoter whose activity is being measured. Promoter activity may be measured directly by determining the amount of RNA transcript produced, for example by Northern blot analysis or indirectly by determining the amount of product coded for by the linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[0179] "Inducible promoter" as used herein refers to a promoter which is induced into activity by the presence or absence of transcriptional regulators, e.g., biotic or abiotic factors. Inducible promoters are useful because the expression of genes operably linked to them can be turned on or off at certain stages of development of an organism or in a particular tissue. Examples of inducible promoters are alcohol-regulated promoters, tetracycline-regulated promoters, steroid regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature regulated promoters and light-regulated promoters. In one embodiment, the inducible promoter is part of a genetic switch. The inducible promoter can be a gene switch ligand inducible promoter. In some cases, an inducible promoter can be a small molecule ligand-inducible two polypeptide ecdysone receptor-based gene switch, such as RHEOSWITCH@ gene switch. In some cases, a gene switch can be selected from ecdysone-based receptor components as described in, but without limitation to, any of the systems described in: PCT/US2001/009050

(WO 2001/070816); U.S. Pat. Nos. 7,091,038; 7,776,587; 7,807,417; 8,202,718; PCT/US2001/030608 (WO 2002/029075); U.S. Pat. Nos. 8,105,825; 8,168,426; PCT/1J52002/005235 (WO 2002/066613); U.S. App. No. 10/468,200 (U.S. Pub. No. 20120167239); PCT/US2002/005706 (WO 2002/066614); U.S. Pat. Nos. 7,531,326; 8,236,556; 8,598,409; PCT/U52002/005090 (WO 2002/066612); U.S. Pat. No. 8,715,959 (U.S. Pub. No. 20060100416); PCT/US2002/005234 (WO 2003/027266); U.S. Pat. Nos. 7,601,508; 7,829,676; 7,919,269; 8,030,067; PCT/U52002/005708 (WO 2002/066615); U.S. App. No. 10/468,192 (U.S. Pub. No. 20110212528); PCT/US2002/005026 (WO 2003/027289); U.S. Pat. Nos. 7,563,879; 8,021,878; 8,497,093; PCT/US2005/015089 (WO 2005/108617); U.S. Pat. No. 7,935,510; 8,076,454; PCT/U52008/011270 (WO 2009/045370); U.S. App. No. 12/241,018 (U.S. Pub. No. 20090136465); PCT/US2008/011563 (WO 2009/048560); U.S. App. No. 12/247,738 (U.S. Pub. No. 20090123441); PCT/US2009/005510 (WO 2010/042189); U.S. App. No. 13/123,129 (U.S. Pub. No. 20110268766); PCT/US2011/029682 (WO 2011/119773); U.S. App. No. 13/636,473 (U.S. Pub. No. 20130195800); PCT/US2012/027515 (WO 2012/122025); and,U.S.Pat.No.9,402,919 each of which is incorporated by reference in its entirety).

[0180] Provided herein are methods comprising administering to a subject at least one non viral vector comprising a polynucleotide encoding a polypeptide sequence described herein comprising at least two functional proteins or portions thereof; at least one promotor; and at least one engineered recombination site; wherein said at least one promoter drives expression of said at least two functional proteins. In some cases, at least one promotor can be constitutive. In some cases, at least one promoter can be tissue-specific. In some cases, at least one promoter can be inducible. In some cases, an inducible promoter is a small molecule ligand-inducible two polypeptide ecdysone receptor-based gene switch. In other cases, a combination of promoters wherein at least one promoter can be inducible and at least one promoter can be activation specific can be utilized.

[0181] An inducible promoter utilizes a ligand for dose-regulated control of expression of said at least two genes. In some cases, a ligand can be selected from a group consisting of ecdysteroid, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazines, oxadiazolines, dibenzoylalkyl cyanohydrazines, N-alkyl-N,N'-diaroylhydrazines, N-acyl-N alkylcarbonylhydrazines, N-aroyl-N-alkyl-N'-aroylhydrazines, arnidoketones, 3,5-di-tert-butyl 4-hydroxy-N-isobutyl-benzamide, 8-0-acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3 sulfate (ECHS), 7-ketocholesterol-3-sulfate, framesol, bile acids, 1,1-biphosphonate esters, juvenile hormone III, RG-115819 (3,5 -Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl propyl)-N'-(2-methyl-3-methoxy-benzoyl)-hydrazide- ), RG-115932 ((R)-3,5-Dimethyl benzoic acid N-(1-tert-butyl-buty 1)-N'-(2-ethy l -3-methoxy-benzoy 1)-hydrazide), and RG 115830 (3,5 -Dimethyl-b enzoic acid N-(1-tert-butyl-buty )-N'-(2-ethyl-3-methoxy-benzoy1) hydrazide), and any combination thereof.

[0182] In some embodiments, a promoter is an inducible promoter. In some embodiments, a promoter is a non-inducible promoter. In some cases, a promoter can be a tissue-specific promoter. Herein "tissue-specific" refers to regulated expression of a gene in a subset of tissues or cell types. In some cases, a tissue-specific promoter can be regulated spatially such that the promoter drives expression only in certain tissues or cell types of an organism. In other cases, a tissue-specific promoter can be regulated temporally such that the promoter drives expression in a cell type or tissue differently across time, including during development of an organism. In some cases, a tissue-specific promoter is regulated both spatially and temporally. In certain embodiments, a tissue-specific promoter is activated in certain cell types either constitutively or intermittently at particular times or stages of the cell type. For example, a tissue-specific promoter can be a promoter that is activated when a specific cell such as a T cell or a NK cell is activated. T cells can be activated in a variety of ways, for example, when presented with peptide antigens by MHC class II molecules or when an engineered T cells comprising an antigen binding polypeptide engages with an antigen. In one instance, such an engineered T cell or NK cell expresses a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0183] In one case, at least one promoter is an engineered promoter or variants thereof. As described herein, the promoter can incorporate minimal promoter sequences from IL-2 and one or more of the following: nuclear factor of activated T-cells (NFAT) response element(s) such as SEQ ID NO 51; NFIL2D response element, NFkB/TCF response element, NFAT/NFIL2B response element or NFIL2A/OCT response element. Examples of response elements are described in Mattila et al., EMBO J. 1990 December; 9(13): 4425-4433; incorporated herein in its entirety.

[0184] In some embodiments, at least one promoter comprises IL-2 core promoter (SEQ ID NO 40). In one embodiment, at least one promoter comprises IL-2 minimal promoter (SEQ ID NO 41). In another embodiment, at least one promoter comprises IL-2 enhancer and promoter variant (SEQ ID NOS 42-43). In yet another embodiment, at least one promoter comprises NF KB binding site (SEQ ID NOS 44-46). In some embodiments, at least one promoter comprises (NF-KB) 1-IL2 promoter variant (SEQ ID NO 47). In some embodiments, at least one promoter comprises (NF-KB) 3-IL2 promoter variant (SEQ ID NO 48). In some embodiments, at least one promoter comprises (NF-KB)-IL2 promoter variant (SEQ ID NO 49). In one embodiments, at least one promoter comprises IX NFAT response elements-IL2 promoter variant (SEQ ID NO

50). In another embodiments, at least one promoter comprises nuclear factor of activated T-cells (NFAT) response element (SEQ ID NO 51). In yet another embodiment, at least one promoter comprises 6X NFAT response elements-IL2 promoter variant (SEQ ID NOS 52-55). In yet another embodiment, at least one promoter comprises 3X NFAT response elements-IL2 promoter variant (SEQ ID NOS 56-57). In some embodiments, at least one promoter comprises human EF1A1 promoter variant (SEQ ID NOS 58-59). In some embodiment, at least one promoter comprises human EF1A1 promoter and enhancer (SEQ ID NO 60). In some embodiments, at least one promoter comprises human UBC promoter (SEQ ID NO 61). In some embodiments, at least one promoter comprises 6 site GAL4-inducible proximal factor binding element (PFB) (SEQ ID NO 62). In some embodiment, at least one promoter comprises synthetic minimal promoter 1 (inducible promoter) (SEQ ID NO 63). Human IL-2 gene and 5' flanking region comprises a nucleotide sequence of SEQ ID NO 95.

[0185] Use of gene switch for ligand inducible control of IL-12 expression described herein can improve the safety profile of IL-12 by for example allowing for regulated expression and improving therapeutic index. However, a condition for ligand dose dependent expression of IL 12 using gene switch(es) is the presence or absence of activator ligand (e.g. veledimex). In certain embodiments, an additional conditional control for induction of IL-12 expression is contemplated. Gene switch components under the control of T cell activated specific promoters are provided. This results in conditional expression (e.g.,T cell activation) of gene switch components necessary for veledimex controlled expression of transgene(s) under control of a gene switch. In some embodiments, this results in preferential expression of cytokines such as IL-12 or IL-15 by tumor specific T cells when veledimex is present and T cells are activated. This may lead to increased localized levels of gene switch controlled transgene expression.

[0186] For example, T cell activation specific expression of gene switch components can be controlled by promoter comprising Nuclear Factor of Activated T-cells (NFAT) response element(s). NFAT transcription factors are key modulators of effector T-cell states. NFATs are early transcriptional checkpoint progressively driving exhaustion. NFATs are quickly activated in T cells following TCR stimulation and form a protein complex with AP-1 induced by appropriate co-stimulation signaling and regulate effector genes and T-cell functions. NFAT response element(s) can be fused with other minimal promoter sequences (e.g. IL2 minimal promoter) to drive expression of transgenes in response to T cell activation.

[0187] Other examples of activation specific promoters include but are not limited to interleukin-2 (IL2) promoter and Programmed Death (PD)-1 (CD279) promoter. Gene switch components can also be conditionally expressed upon immune cell activation by fusing binding sites for other nuclear factors like NF-B of proinflammatory signaling pathway to minimal promoter sequence (e.g. IL2).

[0188] In some embodiments, the promoter comprises NF-KB binding site (SEQ ID NOS 44 46), nuclear factor of activated T cells (NFAT) response element (SEQ ID NO 51), 6 site GAL4 inducible proximal factor binding element (PFB) (SEQ ID NO 62) or synthetic 5' UTR based on RPL6 (SEQ ID NO 64). In certain embodiments, the promoter can be any one or more of: IL-2 core promoter, IL-2 minimal promoter, IL-2 enhancer and promoter variant, (NF-B)-IL2 promoter variant, (NF-x]B) 3-IL2 promoter variant, (NF-xB) 6-IL2 promoter variant, IX NFAT response elements-IL2 promoter variant, 3X NFAT response elements-IL2 promoter variant, 6X NFAT response elements-IL2 promoter variant, human EEF1A1 promoter variant, human EEF1A1 promoter and enhancer, human UBC promoter and synthetic minimal promoter 1. In certain embodiments, the promoter nucleotides comprise disclosed in the table below:

Table 2. Promoter polynucleotide sequences SEQ ID Promoter Polynucleotide Sequence (5' to 3' where applicable) NO ACATTTTGACACCCCCATAATATTTTTCCAGAATTAAC 40 TL-2 core promoter AGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCA CTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTG 41 TL-2 minimal TCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCAC promoter AGTAACCTCAACTCCTG TGATATCTTTTCTGAGTTACTTTTGTATCCCCACCCCC TTAAAGAAAGGAGGAAAAACTGTTTCATACAGAAGG CGTTAATTGCATGAATTAGAGCTATCACCTAAGTGTG GGCTAATGTAACAAAGAGGGATTTCACCTACATCCAT IL-2 enhancer and TCAGTCAGTCTTTGGGGGTTTAAAGAAATTCCAAAGA 42 TL-oeharand GTCATCAGAAGAGGAAAAATGAAGGTAATGTTTTTTC promotervariant AGACTGGTAAAGTCTTTGAAAATATGTGTAATATGTA AAACATTTTGACACCCCCATAATATTTTTCCAGAATTA ACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTAT CACTCTCTTTAATCACTACTCACAGTAACCTCAACTCC TGCCACA TTTTCTGAGTTACTTTTGTATCCCCACCCCCTTAAAGA AAGGAGGAAAAACTGTTTCATACAGAAGGCGTTAATT GCATGAATTAGAGCTATCACCTAAGTGTGGGCTAATG TAACAAAGAGGGATTTCACCTACATCCATTCAGTCAG L-2 enhancer and TCTTTGGGGGTTTAAAGAAATTCCAAAGAGTCATCAG 43 L-2eharand AAGAGGAAAAATGAAGGTAATGTTTTTTCAGACTGGT promotervariant AAAGTCTTTGAAAATATGTGTAATATGTAAAACATTT TGACACCCCCATAATATTTTTCCAGAATTAACAGTAT AAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTC TTTAATCACTACTCACAGTAACCTCAACTCCTGCCAC A

SEQ ID Promoter Polynucleotide Sequence (5' to 3' where applicable) NO AATTGGTCCCATCGAAGAGGGATTTCACCTACATAAT TGGTCCCGGGACATTTTGACACCCCCATAATATTTTTC 47 (Nv-KB)-La CAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGA promotervariant GTTCCCTATCACTCTCTTTAATCACTACTCACAGTAAC CTCAACTCCTG AATTGGTCCCATCGAAGAGGGATTTCACCTACATAAG AGGGATTTCACCTACATAAGAGGGATTTCACCTACAT 48 (NF-KB) -IL2 AATTGGTCCCGGGACATTTTGACACCCCCATAATATT promoter3 variant TTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCA AGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGT AACCTCAACTCCTG AATTGGTCCCATCGAAGAGGGATTTCACCTACATAAG AGGGATTTCACCTACATAAGAGGGATTTCACCTACAT AATTGGTAAGAGGGATTTCACCTACATAAGAGGGATT 49 (N-KB) -IL2 TCACCTACATAAGAGGGATTTCACCTACATAATTGGT promoter6 variant CCCGGGACATTTTGACACCCCCATAATATTTTTCCAG AATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTT CCCTATCACTCTCTTTAATCACTACTCACAGTAACCTC AACTCCTG AATTGGTCCCATCGAATTAGGAGGAAAAACTGTTTCA IX NFAT response TACAGAAGGCGTCAATTGGTCCCGGGACATTTTGACA elements-IL2 CCCCCATAATATTTTTCCAGAATTAACAGTATAAATT promoter variant GCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAA TCACTACTCACAGTAACCTCAACTCCTG TGATATCAATTGGTCCCATCGAATTAGGAGGAAAAAC TGTTTCATACAGAAGGCGTCAATTAGGAGGAAAAACT 3X NFAT response GTTTCATACAGAAGGCGTCAATTAGGAGGAAAAACT 56 elements-IL2 GTTTCATACAGAAGGCGTCAATTGGTCCCGGGACATT promoter variant TTGACACCCCCATAATATTTTTCCAGAATTAACAGTAT AAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTC TTTAATCACTACTCACAGTAACCTCAACTCCTG AATTGGTCCCATCGAATTAGGAGGAAAAACTGTTTCA TACAGAAGGCGTCAATTAGGAGGAAAAACTGTTTCAT 3X NFAT response ACAGAAGGCGTCAATTAGGAGGAAAAACTGTTTCAT 57 elements-IL2 ACAGAAGGCGTCAATTGGTCCCGGGACATTTTGACAC promoter variant CCCCATAATATTTTTCCAGAATTAACAGTATAAATTG CATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAAT CACTACTCACAGTAACCTCAACTCCTG GAATTAGGAGGAAAAACTGTTTCATACAGAAGGCGT CAATTAGGAGGAAAAACTGTTTCATACAGAAGGCGTC AATTAGGAGGAAAAACTGTTTCATACAGAAGGCGTC 6X NFAT response AATTGGTCCCATCGAATTAGGAGGAAAAACTGTTTCA 5XEresponse TACAGAAGGCGTCAATTAGGAGGAAAAACTGTTTCAT 52 promoter vacant ACAGAAGGCGTCAATTAGGAGGAAAAACTGTTTCAT ACAGAAGGCGTCAATTGGTCCCGGGACATTTTGACAC CCCCATAATATTTTTCCAGAATTAACAGTATAAATTG CATCTCTTGTTCAAGAGTTCCCTATCACTCTCCTTAAT CACTACTCACAGTAACCTCAACTCCTG

SEQ ID Promoter Polynucleotide Sequence (5' to 3' where applicable) NO TGATATCGAATTAGGAGGAAAAACTGTTTCATACAGA AGGCGTCAATTAGGAGGAAAAACTGTTTCATACAGA AGGCGTCAATTAGGAGGAAAAACTGTTTCATACAGA AGGCGTCAATTGGTCCCATCGAATTAGGAGGAAAAA 6X NFAT response CTGTTTCATACAGAAGGCGTCAATTAGGAGGAAAAAC 53 elements-IL2 TGTTTCATACAGAAGGCGTCAATTAGGAGGAAAAACT promoter variant GTTTCATACAGAAGGCGTCAATTGGTCCCGGGACATT TTGACACCCCCATAATATTTTTCCAGAATTAACAGTAT AAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTC TTTAATCACTACTCACAGTAACCTCAACTCCTGAATTC CATG GAATTAGGAGGAAAAACTGTTTCATACAGAAGGCGT CAATTAGGAGGAAAAACTGTTTCATACAGAAGGCGTC AATTAGGAGGAAAAACTGTTTCATACAGAAGGCGTC 6X NFAT response AATTGGTCCCATCGAATTAGGAGGAAAAACTGTTTCA 6XEresponse TACAGAAGGCGTCAATTAGGAGGAAAAACTGTTTCAT 54 promoter vacant ACAGAAGGCGTCAATTAGGAGGAAAAACTGTTTCAT ACAGAAGGCGTCAATTGGTCCCGGGACATTTTGACAC CCCCATAATATTTTTCCAGAATTAACAGTATAAATTG CATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAAT CACTACTCACAGTAACCTCAACTCCTG TGATATCGAATTAGGAGGAAAAACTGTTTCATACAGA AGGCGTCAATTAGGAGGAAAAACTGTTTCATACAGA AGGCGTCAATTAGGAGGAAAAACTGTTTCATACAGA 6X NFAT response AGGCGTCAATTGGTCCCATCGAATTAGGAGGAAAAA 5 NEresponse CTGTTTCATACAGAAGGCGTCAATTAGGAGGAAAAAC promoter vacant TGTTTCATACAGAAGGCGTCAATTAGGAGGAAAAACT GTTTCATACAGAAGGCGTCAATTGGTCCCGGGACATT TTGACACCCCCATAATATTTTTCCAGAATTAACAGTAT AAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTC TTTAATCACTACTCACAGTAACCTCAACTCCTG GAGCGTGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAG AGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGG GAGGGGGTCGGCGATTGAACCGGTGCCTAGAGAAGG 58 mother varilt TGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACT GGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTA TATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGC AACGGGTTTGCCGCCAGAACACAG GCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGC ACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGG humanEEF1Al GTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGC 59promotervariant GGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCC prootr vrint GCCT TTTTCCCGAGGGTGGGGGAGAACCGTATATAAG TGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGG TTTGCCGCCAGAACACA humanEEF1Al GAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGA promoter and GGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAA enhancer AGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCA

SEQ ID Promoter Polynucleotide Sequence (5' to 3' where applicable) NO GAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGG GGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGG TGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACT GGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTA TATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGC AACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGT GTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGG CCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGT ACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGG GTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCT TCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCT GGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGC CTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAA ATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAA GATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTG GTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGC CCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGC CTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAG TCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCG CGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCT GGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATG GCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGG AGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCG TCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACG TCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGG AGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAG GCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTG CCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCT CAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGT GTCGTGAG GGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGC CCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAA GGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGC TCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTT AGAACCCCAGTATCAGCAGAAGGACATTTTAGGACG GGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCA GAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTC GGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAAC 61 humanUBCpromoter GCCGATGATTATATAAGGACGCGCCGGGTGTGGCACA GCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTC TTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTAG CGGGCTGCTGGGCTGGGTACGTGCGCTCGGGGTTGGC GAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAA TGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAG ACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGC TTTTTTGTTAGACG 63 synthetic minimal AGGTCTATATAAGCAGAGCTCGTTTAGTGAACCCTCA

SEQ ID Promoter Polynucleotide Sequence (5' to 3' where applicable) NO promoter 1 TTCTGGAGACGGATCCCGAGCCGAGTGTTTTGACCTC CATAGAA

Gene Switch

[0189] Provided herein are gene switch polypeptides, polynucleotides encoding ligand inducible gene switch polypeptides, and methods and systems incorporating these polypeptides and/or polynucleotides.

[0190] The term "gene switch" or "genetic switch" refers to the combination of a response element associated with a promoter, and for instance, an EcR based system which, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are incorporated. Tightly regulated inducible gene expression systems or gene switches are useful for various applications such as gene therapy, large scale production of proteins in cells, cell based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals. Such inducible gene expression systems may include ligand inducible heterologous gene expression systems.

[0191] An early version of EcR-based gene switch used Drosophila melanogasterEcR (DmEcR) and Mus musculus RXR (MmRXR) polypeptides and showed that these receptors in the presence of steroid, ponasteroneA, transactivate reporter genes in mammalian cell lines and transgenic mice (Christopherson et al., 1992; No et al., 1996). Later, Suhr et al., 1998 showed that non-steroidal ecdysone agonist, tebufenozide, induced high level of transactivation of reporter genes in mammalian cells through Bombyx mori EcR (BmEcR) in the absence of exogenous heterodimer partner.

[0192] International Patent Applications No. PCT/US97/05330 (WO 97/38117) and PCT/US99/08381 (W099/58155) disclose methods for modulating the expression of an exogenous gene in which a DNA construct comprising the exogenous gene and an ecdysone response element is activated by a second DNA construct comprising an ecdysone receptor that, in the presence of a ligand therefor, and optionally in the presence of a receptor capable of acting as a silent partner, binds to the ecdysone response element to induce gene expression. In this example, the ecdysone receptor was isolated from Drosophila melanogaster. Typically, such systems require the presence of the silent partner, preferably retinoid X receptor (RXR), in order to provide optimum activation. In mammalian cells, insect ecdysone receptor (EcR) is capable of heterodimerizing with mammalian retinoid X receptor (RXR) and, thereby, be used to regulate expression of target genes or heterologous genes in a ligand dependent manner. International

Patent Application No. PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor isolated from the silk moth Bombyx mori is functional in mammalian systems without the need for an exogenous dimer partner. In some embodiments, the RXR comprises a nucleotide at least 45%, 50%,55%, 60%,65%,70%,75%, 80%,85%,90%,95%, 97%, 98%,99% or 100% identical to SEQ ID NO 69 or a polypeptide at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 182. In some embodiments, the gene switch comprises VP16-linker-RxR, wherein the VP16-linker-RXR comprises a nucleotide at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 70 or a polypeptide at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 183.

[0193] U.S. Pat. No. 6,265,173 discloses that various members of the steroid/thyroid superfamily of receptors can combine with Drosophila melanogaster ultraspiracle receptor (USP) or fragments thereof comprising at least the dimerization domain of USP for use in a gene expression system. U.S. Pat. No. 5,880,333 discloses a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer system used in plants in which the transactivation domain and the DNA binding domain are positioned on two different hybrid proteins. In each of these cases, the transactivation domain and the DNA binding domain (either as native EcR as in International Patent Application No. PCT/US98/14215 or as modified EcR as in International Patent Application No. PCT/US97/05330) were incorporated into a single molecule and the other heterodimeric partners, either USP or RXR, were used in their native state. In some embodiments, the gene switch comprises EcR ligand binding domain - VY variant, wherein the EcR ligand binding domain - VY variant comprises a nucleotide at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 72 or 73 or a polypeptide at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 185 or 186. In other embodiments, the gene switch comprises GAL4-linker-EcR, wherein the GAL4-linker-EcR comprises a nucleotide at least 45%, 50%, 55%, 6 0%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 9 7 %, 9 8 %, 9 9 % or 100% identical to SEQ ID NO 74 or 75 or a polypeptide at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO 187 or 188.

[0194] International Patent Application No. PCT/US01/0905 discloses an ecdysone receptor based inducible gene expression system in which the transactivation and DNA binding domains are separated from each other by placing them on two different proteins results in greatly reduced background activity in the absence of a ligand and significantly increased activity over background in the presence of a ligand. This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the two systems disclosed in applications PCT/US97/05330 and PCT/US98/14215. The two-hybrid system is believed to exploit the ability of a pair of interacting proteins to bring the transcription activation domain into a more favorable position relative to the DNA binding domain such that when the DNA binding domain binds to the DNA binding site on the gene, the transactivation domain more effectively activates the promoter (see, for example, U.S. Pat. No. 5,283,173). The two-hybrid gene expression system comprises two gene expression cassettes; the first encoding a DNA binding domain fused to a nuclear receptor polypeptide, and the second encoding a transactivation domain fused to a different nuclear receptor polypeptide. In the presence of ligand, it is believed that a conformational change is induced which promotes interaction of the first polypeptide with the second polypeptide thereby resulting in dimerization of the DNA binding domain and the transactivation domain. Since the DNA binding and transactivation domains reside on two different molecules, the background activity in the absence of ligand is greatly reduced.

[0195] Another surprising discovery was that certain modifications of the two-hybrid system could also provide improved sensitivity to non-steroidal ligands for example, diacyhydrazines, when compared to steroidal ligands for example, ponasterone A ("PonA") or muristerone A ("MurA"). That is, when compared to steroids, the non-steroidal ligands provided higher gene transcription activity at a lower ligand concentration. Furthermore, the two-hybrid system avoids some side effects due to overexpression of RXR that can occur when unmodified RXR is used as a switching partner. In a preferred two-hybrid system, native DNA binding and transactivation domains of EcR or RXR are eliminated and as a result, these hybrid molecules have less chance of interacting with other steroid hormone receptors present in the cell, thereby resulting in reduced side effects.

[0196] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily and is classified into subfamily 1, group H (referred to herein as "Group H nuclear receptors"). The members of each group share 40-60% amino acid identity in the E (ligand binding) domain (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97: 161-163). In addition to the ecdysone receptor, other members of this nuclear receptor subfamily 1, group H include: ubiquitous receptor (UR), Orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXR interacting protein-15 (RIP-15), liver x receptor (LXR), steroid hormone receptor like protein (RLD-1), liver x receptor (LXR), liver x receptor a (LXRa), farnesoid x receptor (FXR), receptor interacting protein 14 (RIP-14), and farnesol receptor (HRR-1).

[0197] In some cases, an inducible promoter can be a small molecule ligand-inducible two polypeptide ecdysone receptor-based gene switch, such as Intrexon Corporation's RHEOSWITCH© gene switch. In some cases, a gene switch can be selected from ecdysone based receptor components as described in, but without limitation to, any of the systems described in: PCT/US2001/009050 (WO 2001/070816); U.S. Pat. Nos. 7,091,038; 7,776,587; 7,807,417; 8,202,718; PCT/US2001/030608 (WO 2002/029075); U.S. Pat. Nos. 8,105,825; 8,168,426; PCT/1J52002/005235 (WO 2002/066613); U.S. App. No. 10/468,200 (U.S. Pub. No. 20120167239); PCT/US2002/005706 (WO 2002/066614); U.S. Pat. Nos. 7,531,326; 8,236,556; 8,598,409; PCT/U52002/005090 (WO 2002/066612); U.S. Pat. No. 8,715,959 (U.S. Pub. No. 20060100416); PCT/US2002/005234 (WO 2003/027266); U.S. Pat. Nos. 7,601,508; 7,829,676; 7,919,269; 8,030,067; PCT/U52002/005708 (WO 2002/066615); U.S. App. No. 10/468,192 (U.S. Pub. No. 20110212528); PCT/US2002/005026 (WO 2003/027289); U.S. Pat. Nos. 7,563,879; 8,021,878; 8,497,093; PCT/US2005/015089 (WO 2005/108617); U.S. Pat. No. 7,935,510; 8,076,454; PCT/U52008/011270 (WO 2009/045370); U.S. App. No. 12/241,018 (U.S. Pub. No. 20090136465); PCT/US2008/011563 (WO 2009/048560); U.S. App. No. 12/247,738 (U.S. Pub. No. 20090123441); PCT/US2009/005510 (WO 2010/042189); U.S. App. No. 13/123,129 (U.S. Pub. No. 20110268766); PCT/US2011/029682 (WO 2011/119773); U.S. App. No. 13/636,473 (U.S. Pub. No. 20130195800); PCT/US2012/027515 (WO 2012/122025); and,U.S.Pat.No.9,402,919 each of which is incorporated by reference in its entirety.

[0198] Provided are systems for modulating the expression of a heterologous gene and an interleukin in a host cell, comprising polynucleotides expressing gene-switch polypeptides disclosed herein. Various structural components of non-limiting exemplary ligand-inducible gene switch vector system under the control of constitutive or inducible promoters are shown in FIG. 22.

[0199] In some embodiments, the expression cassette of the gene switch vector system is XON-64 and has a sequence as shown in SEQ ID NO: 131. In some embodiments, the expression cassette of the gene switch vector system is XON-30 and has a sequence as shown in SEQ ID NO: 132. In some embodiments, the expression cassette of the gene switch vector system is XON-59 and has a sequence as shown in SEQ ID NO: 133. In some embodiments, the expression cassette of the gene switch vector system is XON-60 and has a sequence as shown in SEQ ID NO: 134. In some embodiments, the expression cassette of the gene switch vector system is XON-61 and has a sequence as shown in SEQ ID NO: 135. In some embodiments, the expression cassette of the gene switch vector system is XON-62 and has a sequence as shown in SEQ ID NO: 136.

[0200] In some embodiments are systems for modulating the expression of a heterologous gene and a cytokine in a host cell, comprising a first gene expression cassette comprising a first polynucleotide encoding a first polypeptide; a second gene expression cassette comprising a second polynucleotide encoding a second polypeptide; and a ligand; wherein said first and second polypeptides comprise one or more of: (i) a transactivation domain; (ii) a DNA-binding domain; and (iii) a ligand binding domain; (iv) said heterologous gene; and (vi) said cytokine such that upon contacting said host cell with said first gene expression cassette and said second gene expression cassette in the presence of said ligand, said heterologous gene and said cytokine are expressed in said host cell. In some cases, the heterologous gene comprises an antigen binding polypeptide described herein. In some cases, the antigen binding polypeptide may be a CAR described herein, for instance, a CAR that binds at least one of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR,MUC-1, MUC-16, MAGE-Al, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2.

Cytokines

[0201] Provided herein are polynucleotides encoding gene-switch polypeptides and a cytokine, or variant or derivative thereof, and methods and systems incorporating the same. Cytokine is a category of small proteins between about 5-20 kDa that are involved in cell signaling. In some instances, cytokines include chemokines, interferons, interleukins, colony-stimulating factors or tumor necrosis factors. In some embodiments, chemokines play a role as a chemoattractant to guide the migration of cells, and is classified into four subfamilies: CXC, CC, CX3C, and XC. Exemplary chemokines include chemokines from the CC subfamily: CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13,CCL14,CCL15,CCL16,CCL17,CCL18,CCL19,CCL20,CCL21,CCL22,CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28; the CXC subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17; the XC subfamily: XCL1 and XCL2; and the CX3C subfamily CX3CL1.

[0202] Interferons (IFNs) comprise interferon type I (e.g. IFN-a, IFN-, IFN-g, IFN-K, and IFN-o), interferon type II (e.g. IFN-y), and interferon typeIII. In some embodiments, IFN-a is further classified into about 13 subtypes including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[0203] Interleukins are expressed by leukocytes or white blood cells and they promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Exemplary interleukines include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36.

[0204] In some embodiments, an interleukin comprises mbIL-15. In some embodiments, a mbIL-15 is a membrane-bound chimeric IL-15 which can be co-expressed with a modified effector cell described herein. In some embodiments, the mbIL-15 comprises a full-length IL-15 (e.g., a native IL-15 polypeptide) or fragment or variant thereof, fused in frame with a full length IL-15Ra, functional fragment or variant thereof In some cases, the IL-15 is indirectly linked to the IL-15Ra through a linker. In some instances, the mbIL-15 is as described in Hurton et al., "Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor specific T cells," PNAS 2016.

[0205] Tumor necrosis factors (TNFs) are a group of cytokines that modulate apoptosis. In some instances, there are about 19 members within the TNF family, including, not limited to, TNFa, lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT-beta), T cell antigen gp39 (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and TNF-related apoptosis inducing ligand (TRAIL).

[0206] Colony-stimulating factors (CSFs) are secreted glycoproteins that interact with receptor proteins on the surface of hemopoietic stem cells, which subsequently modulates cell proliferation and differentiation into specific kind of blood cells. In some instances, a CSF comprises macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) or promegapoietin.

[0207] In some embodiments, the cytokine is a membrane-bound cytokine, which is co expressed with a chimeric antigen receptor described herein.

[0208] In some embodiments, one or more methods described herein further comprise administration of a cytokine. In some instances, the cytokine comprises a chemokine, an interferon, an interleukin, a colony-stimulating factor or a tumor necrosis factor. In some instances, one or more methods described herein further comprise administration of a cytokine selected from a chemokine, an interferon, an interleukin, a colony-stimulating factor or a tumor necrosis factor. In some instances, one or more methods described herein further comprise administration of a cytokine selected from IL2, IL7, IL12, IL15, IL21, IFNy or TNF-a.

[0209] In some cases, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variant or combination thereof In some cases, the cytokine is an interleukin. In some cases, the interleukin is at least one of IL-12, IL-2, IL-15, IL-21, and functional variants and fragments thereof. In some embodiments, the cytokine is at least one of IL-1, IL-2, IL-15, IL-12, IL-21, a fusion of IL-15, IL-15Ra, or an IL-15 variant. In one embodiment, the cytokine is a variant of IL-15 (SEQ ID NO 90; SEQ ID NO 203). In one embodiment, the cytokine comprises IL-15 receptor alpha (SEQ ID NO 91; SEQ ID NO 204). In some embodiments, the cytokines can be membrane bound or secreted. In other embodiments, the cytokines can be intracellular. The interleukin may comprise membrane bound IL-15 (mbIL 15), a fusion of IL-15 and IL-15Ra (SEQ ID NO 91; SEQ ID NO 204) or an IL-15 (SEQ ID NO

90; SEQ ID NO 203) variant. In some embodiments, a mbIL-15 is a membrane-bound chimeric IL-15 which can be co-expressed with a modified effector cell described herein. In some embodiments, the mbIL-15 comprises a full-length IL-15 (e.g., a native IL-15 polypeptide) or fragment or variant thereof, fused in frame with a full length L-15Ra, functional fragment or variantthereof In some cases, the IL-15 is indirectly linked to the IL-15Ra through a linker. In some embodiments, the mbIL-15 comprises a signal peptide (SEQ ID NO 92; SEQ ID NO 205). In some instances, the mbIL-15 is as described in Hurton et al., "Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells," PNAS 2016. In another aspect, the interleukin can comprise IL-12. In some embodiments, IL-12 can be a single chain IL-12 (scIL-12), protease sensitive IL-12\, destabilized IL-12, membrane bound IL-12 or intercalated IL-12. In some embodiments, IL-12 is murine IL-12 subunit beta (p40) (SEQ ID NO 206). In some embodiments, IL-12 is murine IL-12 subunit alpha (p35) (SEQ ID NO 207). In some embodiments, IL-12 is murine single chain L-12 (p40-linker-p35) (SEQ ID NO 93; SEQ ID NO 208). In some embodiments, IL-12 is human single chain IL-12 (p40-linker p35) (SEQ ID NO 94; SEQ ID NO 209). In certain embodiments, IL-12 is single chain IL-12. In some instances, the IL-12 variants are as described in W02015/095249, W02016/048903, and W02017/062953, all of which are incorporated by reference in their entireties.

[0210] Provided herein are polynucleotides encoding gene switch polypeptides, wherein said gene switch polypeptides comprise: a) a first gene switch polypeptide comprising a DNA-binding domain fused to a nuclear receptor ligand binding domain, and b) a second gene switch polypeptide comprising a transactivation domain fused to a nuclear receptor ligand binding domain, wherein the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In some cases, the linker may be a linker described herein, for instance GSG linker, furinlink, a 2A linker such as F/T2A, T2A, p2A, GSG-p2A, variants and derivatives thereof. In other instances, the linker may be an IRES. In some embodiments, the IRES is EMCV IRES or 2xRbm3 IRES. Exemplary IRES sequences can be found in SEQ ID NO: 18 and 19. In certain cases, a polynucleotide encoding 2xRbm3 RES a can be or can be about at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 19. In certain cases, a polynucleotide encoding EMCV RES a can be or can be about at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18.

[0211] In some cases, the DNA binding domain (DBD) comprises a DBD described herein, for instance at least one of GAL4 (GAL4 DBD) (SEQ ID NO 71; SEQ ID NO 184), a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, and a yeast DBD. The transactivation domain may comprise a transactivation domain described herein, for instance one of a VP16 transactivation domain (SEQ ID NO 68; SEQ ID NO 181), a p53 transactivation domain and a B42 acidic activator transactivation domain. The Nuclear receptor ligand binding domain may comprise at least one of a ecdysone receptor (EcR), a ubiquitous receptor, an orphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor p, a steroid hormone receptor like protein, a liver X receptor, a liver X receptor a, a farnesoid X receptor, a receptor interacting protein 14, and a famesol receptor.

[0212] In some cases, the gene switch polypeptides connected by a polypeptide linker or ribosome-skipping sequence exhibit improved dose-dependent ligand-inducible control of gene expression compared to a ligand-inducible gene switch wherein the gene switch polypeptides are connected by non-coding sequences, such as an IRES. In some cases, the gene switch polypeptides connected by a 2A linker may exhibit improved dose-dependent ligand-inducible control of heterologous gene expression compared to a gene switch wherein said gene switch polypeptides are separated by an IRES.

[0213] The polypeptides and polynucleotides as described herein can be expressed in an engineered cell. Herein an engineered cell is a cell which has been modified from its natural or endogenous state. An example of an engineered cell is a cell described herein which has been modified (e.g., by transfection of a polynucleotide into the cell) to encode for example, gene switch polypeptides, gene of interest (GOI), cell tags, heterologous genes and any other polypeptides and polynucleotides described herein.

Ligands

[0214] In some embodiments, a ligand used for inducible gene switch regulation can be selected from any of, but without limitation to, following: N-[(1R)-1-(1,1-dimethylethyl)butyl] N'-(2-ethyl-3-methoxybenzoyl)-3,5-dimethylbenzohydrazide (also referred to as veledimex), (2S,3R,5R,9R,1OR,13R,14S,17R)-17- [(2S,3R)-3,6-dihydroxy-6-methylheptan- 2-yl]-2,3,14 trihydroxy-10,13-dimethyl- 2,3,4,5,9,11,12,15,16,17-decahydro- 1H-cyclopenta[a]phenanthren 6-one; N'-(3,5-Dimethylbenzoyl)-N'-[(3R)-2,2-dimethyl-3-hexanyl]-2-ethyl-3 methoxybenzohydrazide; 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5 dimethyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-Methyl-2,3-dihydro benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2 dimethyl-propyl)-hydrazide; 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1 tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl)-hydrazide; 5-Methyl-2,3-dihydro-benzo[1,4]dioxine 6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide; 5 Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethyl-benzoyl)-N'-(1-ethyl 2,2-dimethyl-propyl)-hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N' (3,5-dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-Ethyl-2,3 dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl) hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(-tert-butyl-butyl)-N' (3,5-dimethoxy-4-methyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2 dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5 Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5 Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methoxy-2-methyl-benzoyl) hydrazide; 3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-ethyl-3-methoxy benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N' (2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2 ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 2-Methoxy-nicotinic acid N-(1-tert-butyl pentyl)-N'-(4-ethyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(2,2-dimethyl-1-phenyl propyl)-N'-(4-ethyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-pentyl)-N' (3-methoxy-2-methyl-benzoyl)-hydrazide; and 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide.

[0215] In some cases, a ligand used for dose-regulated control of ecdysone receptor-based inducible gene switch can be selected from any of, but without limitation to, an ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazines such as those disclosed in U.S. Pat. Nos. 6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S. Published Application Nos. 2005/0209283 and 2006/0020146; oxadiazolines as described in U.S. Published Application No. 2004/0171651; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No. 461,809; N-alkyl-N,N'-diaroylhydrazines such as those disclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application No. 234,994; N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U.S. Pat. No. 4,985,461; arnidoketones such as those described in U.S. Published Application No. 2004/0049037; each of which is incorporated herein by reference and other similar materials including 3,5-di-tert-butyl 4-hydroxy-N-isobutyl-benzamide, 8-0-acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3 sulfate (ECHS), 7-ketocholesterol-3-sulfate, framesol, bile acids, 1,1-biphosphonate esters, juvenile hormone III, and the like. Examples of diacylhydrazine ligands useful in the present disclosure include RG-115819 (3,5 -Dimethyl-benzoic acid N-(1-ethy l-2,2-dimethyl-propy l) N'-(2-methyl-3-methoxy-benzoy1)-hydrazide- ), RG-115932 ((R)-3,5-Dimethyl-benzoic acid N (1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-115830 (3,5 Dimethyl-b enzoic acid N-(1-tert-butyl-buty)-N'-(2-ethyl-3-methoxy-benzoy)-hydrazide). See, e.g., U.S. patent application Ser. No. 12/155,111, and PCT Appl. No. PCT/US2008/006757, both of which are incorporated herein by reference in their entireties.

Antigen Binding Polypeptides

[0216] Polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4 nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, 3-phenylserine 3 hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6 hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, a, diaminobutyric acid, a,p-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0217] "Antibody" as used herein refers to monoclonal or polyclonal antibodies. The term "monoclonal antibodies," as used herein, refers to antibodies that are produced by a single clone

of B-cells and bind to the same epitope. In contrast, "polyclonal antibodies" refer to a population of antibodies that are produced by different B-cells and bind to different epitopes of the same antigen. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have a similar general structure, with each region comprising four framework regions, whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen binding.

[0218] "Antigen recognition moiety" or "antibody recognition domain" refers to a molecule or portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, antibody like molecule or fragment thereof and the antigen is a tumor antigen or an infectious disease antigen.

[0219] "Antibody like molecules" may be for example proteins that are members of the Ig superfamily which are able to selectively bind a partner. MC molecules and T cell receptors are such molecules. In one embodiment the antibody-like molecule is a TCR. In one embodiment the TCR has been modified to increase its MC binding affinity.

[0220] The terms "fragment of an antibody," "antibody fragment," "functional fragment of an antibody," "antigen-binding portion" or their grammatical equivalents are used interchangeably herein to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the stalk region; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)) and (v) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites. Antibody fragments are known in the art and are described in more detail in, e.g., U.S. Pat. No.

8,603,950. Other antibody fragments can include variable fragments of heavy chain antibodies (VHH).

[0221] The term "functional portion," when used in reference to a CAR, refers to any part or fragment of the CAR of the present disclosure, which part or fragment retains the biological activity of the CAR of which it is a part (the parent CAR). In reference to a nucleic acid sequence encoding the parent CAR, a nucleic acid sequence encoding a functional portion of the CAR can encode a protein comprising, for example, about 10%, 25%, 30%, 50%, 6 8 %, 80%, 90%, 95%, or more, of the parent CAR.

[0222] The term "functional variant," as used herein, refers to a polypeptide, or a protein having substantial or significant sequence identity or similarity to the reference polypeptide, and retains the biological activity of the reference polypeptide of which it is a variant. In some embodiments, a functional variant, for example, comprises the amino acid sequence of the reference protein with at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 conservative amino acid substitutions. Functional variants encompass, for example, those variants of the CAR described herein (the parent CAR) that retain the ability to recognize target cells to a similar extent, the same extent, or to a higher extent, as the parent CAR. In reference to a nucleic acid sequence encoding the parent CAR, a nucleic acid sequence encoding a functional variant of the CAR can be for example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical to the nucleic acid sequence encoding the parent CAR.

[0223] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, for example, lysine for arginine and vice versa such that a positive charge may be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH 2 can be maintained. Alternatively or additionally, the functional variants can comprise the amino acid sequence of the reference protein with at least one non conservative amino acid substitution.

[0224] The term "non-conservative mutations" involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR.

[0225] In some embodiments, the antigen binding moiety of a CAR described herein is specific to or binds CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-Al, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2. In embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising a variable domain light chain (VL) and variable domain heavy chain (VH) of a target antigen specific monoclonal antibody joined by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv is humanized. In some embodiments, the antigen binding moiety may comprise VH and VL that are directionally linked, for example, from N to C terminus, VH-linker-VL or VL-linker-VH. In some instances, the antigen binding domain recognizes an epitope of the target. In some embodiments, described herein include a CAR or a CAR-T cell, in which the antigen binding domain comprises a F(ab')2, Fab', Fab, Fv, or scFv.

[0226] In one embodiment, the antigen binding moiety of a CAR described herein is specific to CD19. In one embodiment, the antigen binding moiety of a CAR described herein is specific to CD33. In another embodiment, the antigen binding moiety of a CAR described herein is specific to BCMA. In yet another embodiment, the antigen binding moiety of a CAR described herein is specific to CD44. In some embodiments, the antigen binding moiety of a CAR described herein is specific to a-Folate receptor. In some embodiments, the antigen binding moiety of a CAR described herein is specific to CAIX. In one embodiment, the antigen binding moiety of a CAR described herein is specific to CD30. In some embodiments, the antigen binding moiety of a CAR described herein is specific to ROR. In one embodiment, the antigen binding moiety of a CAR described herein is specific to CEA. In some embodiments, the antigen binding moiety of a CAR described herein is specific to EGP-2. In one embodiment, the antigen binding moiety of a CAR described herein is specific to EGP-40. In another embodiment, the antigen binding moiety of a CAR described herein is specific to HER2. In yet another embodiment, the antigen binding moiety of a CAR described herein is specific to HER3. In yet another embodiment, the antigen binding moiety of a CAR described herein is specific to Folate-binding protein. In some embodiments, the antigen binding moiety of a CAR described herein is specific to GD2. In some embodiments, the antigen binding moiety of a CAR described herein is specific to GD3. In one embodiment, the antigen binding moiety of a CAR described herein is specific to IL-13R-a2. In one embodiment, the antigen binding moiety of a CAR described herein is specific to KDR. In one embodiment, the antigen binding moiety of a CAR described herein is specific to EDB-F. In another embodiment, the antigen binding moiety of a CAR described herein is specific to mesothelin. In yet another embodiment, the antigen binding moiety of a CAR described herein is specific to CD22. In one embodiment, the antigen binding moiety of a CAR described herein is specific to EGFR. In one embodiment, the antigen binding moiety of a CAR described herein is specific to MUC-1. In one embodiment, the antigen binding moiety of a CAR described herein is specific to MUC-16. In one embodiment, the antigen binding moiety of a CAR described herein is specific to MAGE-Al. In some embodiments, the antigen binding moiety of a CAR described herein is specific to h5T4. In some embodiments, the antigen binding moiety of a CAR described herein is specific to PSMA. In another embodiment, the antigen binding moiety of a CAR described herein is specific to TAG-72. In yet one embodiment, the antigen binding moiety of a CAR described herein is specific to EGFRvIII. In another embodiment, the antigen binding moiety of a CAR described herein is specific to CD123. In yet embodiment, the antigen binding moiety of a CAR described herein is specific to VEGF-R2.

Chimeric Antigen Receptors (CARs)

[0227] In some embodiments, described herein includes a polynucleotide which encodes a chimeric receptor expressed on the surface of the cell. In some instances, the chimeric receptor comprises an antigen binding region that enables recognition and binding to an antigen, for instance, a tumor antigen such as a tumor-associated antigen or a tumor-specific antigen. In some instances, the antigen binding region comprises an antibody or binding fragment, for example, an Fab, an Fab', an F(ab')2, an F(ab')3, an scFv, an sc(Fv)2, a dsFv, a diabody, a minibody, and a nanobody or binding fragments thereof. In some cases, the antigen binding region comprises an scFv. In some cases, the chimeric receptor comprises an scFv (e.g., a chimeric antigen receptor (CAR)). In some instances, the chimeric antigen receptor comprises a pattern-recognition receptor. In other cases, the chimeric receptor comprises an engineered T-cell receptor (TCR).

[0228] The terms "chimeric antigen receptor (CAR)", "artificial T cell receptor", "chimeric T cell receptor" or "chimeric immunoreceptor" as used herein refer to an engineered receptor which grafts an exogenous specificity onto an immune effector cell. In some instances, a CAR comprises an extracellular domain (ectodomain) that comprises an antigen binding domain, a stalk region, a transmembrane domain and an intracellular (endodomain) domain. In some instances, the intracellular domain further comprises one or more intracellular signaling domains. In some instances, a CAR described herein comprises an antigen binding domain, a stalk region, a transmembrane domain, one or more costimulatory domains, and a signaling domain for T-cell activation.

[0229] In embodiments, the CAR of the present disclosure comprises a target-specific binding element otherwise referred to as an antigen-binding moiety. In embodiments, the CAR of the present disclosure is engineered to target a tumor antigen of interest by way of engineering a desired antigen-binding moiety that specifically binds to an antigen on a tumor cell. In the context of the present disclosure, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder," refers to antigens that are common to specific hyperproliferative disorders such as cancer.

[0230] An antigen binding domain can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen binding fragments thereof. A complementarity determining region (CDR) is a short amino acid sequence found in the variable domains of antigen receptor (e.g., immunoglobulin and T-cell receptor) proteins that complements an antigen and therefore provides the receptor with its specificity for that particular antigen. Each polypeptide chain of an antigen receptor can contain three CDRs (CDR1, CDR2, and CDR3). In some instances, an antigen binding domain comprises F(ab')2, Fab', Fab, Fv, or scFv. In some cases, an antigen binding domain is a scFv. In some cases, an antigen binding domain is a Fab. In some cases, an antigen binding domain is a Fab'. In some cases, an antigen binding domain is F(ab')2. In some cases, an antigen binding domain is a Fv.

[0231] In some embodiments, a CAR described herein comprises an antigen binding domain that binds to an epitope on CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-Al, MUC16, h5T4, PSMA, TAG 72, EGFRvIII, CD123 and VEGF-R2. In some embodiments, a CAR described herein comprises an antigen binding domain that binds to an epitope on CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-Al, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2. In some embodiments, a CAR described herein comprises an antigen binding domain that binds to an epitope on CD19, CD33 or EGFRvIII. In some instances, a CAR described herein comprises an antigen binding domain that binds to an epitope on CD19. In some cases, a CAR described herein comprises an antigen binding domain that binds to an epitope on CD33. In further embodiments, a CAR or a chimeric receptor or antigen binding polypeptide described herein comprises an autoantigen or an antigen binding region that binds to an epitope onHLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1, or collagen type II.

[0232] In another embodiment, a CAR described herein is a EGFRvIII specific CAR. "EGFRvIII", "EGFR variant III", "EGFR type III mutant", "EGFR.D2-7" or "de2-7EGFR" is a mutated form of epidermal growth factor receptor (EGFR; ErbB-1; HERI), a transmembrane protein that is a receptor for members of the epidermal growth factor (EGF) family of extracellular protein ligands in human and non-human subjects. EGFRvIII is characterized by a deletion of exons 2-7 of the wild type EGFR gene, which results in an in-frame deletion of 267 amino acids in the extracellular domain of the full length wild type EGFR protein. EGFRvIII also contains a novel glycine residue inserted at the fusion junction. The truncated receptor EGFRvIII is unable to bind any known EGFR ligand; however, it shows constitutive tyrosine kinase activity. This constitutive activation is important to its pro-oncogenic effect. A kinase deficient EGFRvIII is unable to confer a similar oncogenic advantage. EGFRvIII is highly expressed in glioblastoma (GBM) and can be detected in some other solid tumor types but not in normal tissues.

[0233] In some embodiments, the antigen binding moiety of a CAR described herein is specific to EGFRvIII (EGFRvIII CAR). The EGFRvIII-specific CAR, when expressed on the cell surface, redirects the specificity of T cells to human EGFRvIII. In embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising a variable domain light chain (VL) and variable domain heavy chain (VH) of a target antigen specific monoclonal anti-EGFRvIII antibody joined by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv is murine MR1 IgG. In some embodiments, the scFv is anti EGFRvIII scFv clone MIR (SEQ ID NO 115; SEQ ID NO 229), anti-EGFRvIII scFv clone MIR1-1 (SEQ ID NO 116; SEQ ID NO 230), anti-EGFRvIII scFv clone huMR1-1 (SEQ ID NO 117; SEQ ID NO 231), anti-EGFRvIII scFv clone huMR1-2 (SEQ ID NO 118; SEQ ID NO 232). In some embodiments, the antigen binding moiety may comprise VH and VL that are directionally linked, for example, from N to C terminus, VH-linker-VL or VL-linker-VH.

[0234] In embodiments, a CAR described herein comprises an antigen-binding moiety comprising a VL polypeptide having at least 90%, 91%, 9 2 %, 93%, 9 4 %, 9 5 %, 9 6 %, 9 7 %, 9 8 %,

99% or 100% identity with the amino acid sequence of SEQ ID NO 221 (anti-EGFRvIII clone MIR VL). In embodiments, a CAR described herein comprises an antigen-binding moiety comprising a VH polypeptide having at least 90%, 91%, 9 2 %, 9 3 %, 9 6 %, 9 7 %, 9 8 %, 94%, 95%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 220 (anti-EGFRvIII clone

MR1 VH). In embodiments, a CAR described herein comprises an antigen-binding moiety comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 223 (anti-EGFRvIII clone MR1-1 VL). In embodiments, a CAR described herein comprises an antigen-binding moiety comprising a VH polypeptide having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 9 6 %, 9 8 %, 97%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 222 (anti-EGFRvIII clone MR1-1 VH).

[0235] In embodiments, a CAR described herein comprises an antigen-binding moiety comprising a VL polypeptide having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 9 6 %, 9 8 %, 97%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 225 (anti-EGFRvIII clone humMIR-1 VL). In embodiments, a CAR described herein comprises an antigen-binding moiety comprising a VH polypeptide having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 9 6 %, 9 8 %, 97%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 224 (anti-EGFRvIII clone humMR1-1 VH). In embodiments, a CAR described herein comprises an antigen-binding moiety comprising a VL polypeptide having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 9 6 %, 9 8 %, 97%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 227 (anti-EGFRvIII clone humMR1-2 VL). In embodiments, a CAR described herein comprises an antigen-binding moiety comprising a VH polypeptide having at least 90%, 91%, 9 2 %, 9 6 %, 9 8 %, 93%, 94%, 95%, 97%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 226 (anti-EGFRvIII clone humM R1-2 VH).

[0236] In one embodiment, a CAR described herein is a CD19 specific CAR. "CD19", cluster of differentiation 19 or B-lymphocyte antigen CD19, is a protein that in human is encoded by the CD19 gene. The CD19 gene encodes a cell surface molecule that assembles with the antigen receptor of B lymphocytes in order to decrease the threshold for antigen receptor-dependent stimulation. CD19 is expressed on follicular dendritic cells and B cells. In fact, it is present on B cells from earliest recognizable B-lineage cells during development to B-cell blasts but is lost on maturation to plasma cells. It primarily acts as a B cell co-receptor in conjunction with CD21 and CD81. Upon activation, the cytoplasmic tail of CD19 becomes phosphorylated, which leads to binding by Src-family kinases and recruitment of PI-3 kinase. As on T cells, several surface molecules form the antigen receptor and form a complex on B lymphocytes. The (almost) B cell specific CD19 phosphoglycoprotein is one of these molecules. The others are CD21 and CD81. These surface immunoglobulin (slg)-associated molecules facilitate signal transduction. On B cells, anti-immunoglobulin antibody mimicking exogenous antigen causes CD19 to bind to sIg and internalize with it. The reverse process has not been demonstrated, suggesting that formation of this receptor complex is antigen-induced. This molecular association has been confirmed by chemical studies.

[0237] In yet another embodiment, a CAR descried herein is a CD33 specific CAR. "CD33", Siglec-3, sialic acid binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, or p67 is a 67kDa single pass transmembrane glycoprotein and is a member of the sialic acid-binding immunoglobulin-like lectins (Siglecs) super-family. CD33 is characterized by a V-set Ig-like domain responsible for sialic acid binding and a C2-set Ig-like domain in its extracellular domain. Alternative splicing of CD33 mRNA leads to a shorter isoform (CD33m) lacking the V set Ig-like domain as well as the disulfide bond linking the V- and C2-set Ig-like domains. In healthy subjects, CD33 is primarily expressed as a myeloid differentiation antigen found on normal multipotent myeloid precursors, unipotent colony-forming cells, monocytes and maturing granulocytes. CD33 is expressed on more than 80% of myeloid leukemia cells but not on normal hematopoietic stem cells or mature granulocytes (Andrews, R. et al., The L4F3 antigen is expressed by unipotent and multipotent colony-forming cells but not by their precursors, Blood, 68(5):1030-5 (1986)). CD33 has been reported to be expressed on malignant myeloid cells, activated T cells and activated NK cells and is found on at least a subset of blasts in the vast majority of AIL patients (Pollard, J. et al., Correlation of CD33 expression level with disease characteristics and response to gemtuzumab ozogamicin containing chemotherapy in childhood AML, Blood, 119(16):3705-11 (2012)). In addition to broad expression on ANIL blasts, CD33 may be expressed on stem cells underlying AML.

[0238] In embodiments, the antigen binding moiety of a CAR described herein is specific to CD33 (CD33 CAR). The CD33-specific CAR, when expressed on the cell surface, redirects the specificity of T cells to human CD33. In embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising a variable domain light chain (VL) and variable domain heavy chain (VH) of a target antigen specific monoclonal anti-CD33 antibody joined by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv are M195, m2H12, DRB2, and/or My9-6. In embodiments, the scFv is humanized, for example, hM195. In some embodiments, the antigen binding moiety may comprise VH and VL that are directionally linked, for example, from N to C terminus, VH-linker-VL or VL-linker-VH. In some embodiments, the CD33 antigen binding domain comprises a polypeptide having at least 9 0% , 9 1 % , 9 2 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 214 (hM195 VL). In some embodiments, the CD33 antigen binding domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 215 (hM195 VH). In some embodiments, the CD33 antigen binding domain comprises a polypeptide having at least

90%,91%,92%,93%, 94%, 95%, 96%,97%, 98%,99% or 100% identity with the amino acid sequence of SEQ ID NO 216 (hM195 scFv with linker).

[0239] In some embodiments, the polynucleotides, polypeptides and methods described herein can be used for the treatment of a hyperproliferative disease, such as a cancer, an autoimmune disease or for the treatment of an infection, such as a viral, bacterial or parasitic infection. In some aspects, the antigen is an antigen that is elevated in cancer cells, in autoimmune cells or in cells that are infected by a virus, bacteria or parasite. Pathogens that may be targeted include, without limitation, Plasmodium, trypanosome, Aspergillus, Candida, Hepatitis A, Hepatitis B, Hepatitis C, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogens. Autoimmune diseases can include graft-versus-host disease, rheumatoid arthritis, lupus, celiac disease, Crohn's disease, Sjogren Syndrome, polymyalgia rheumatic, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, Type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, or autoimmune uveitis.

[0240] The pathogen recognized by a CAR may be essentially any kind of pathogen, but in some embodiments the pathogen is a fungus, bacteria, or virus. Exemplary viral pathogens include those of the families of Adenoviridae, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), JC virus, BK virus, HPV, HSV, HHV family of viruses, Hepatitis family of viruses, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella. In some embodiments the pathogen receptor Dectin-1 may be used to generate a CAR that recognizes the carbohydrate structure on the cell wall of fungi such as Aspergillus. In another embodiment, CARs can be made based on an antibody recognizing viral determinants (e.g., the glycoproteins from CMV and Ebola) to interrupt viral infections and pathology.

[0241] In some embodiments, a "stalk", "stalk region" or "stalk domain", which encompasses the terms "spacer", "spacer region" or "spacer domain" or "hinge", "hinge region" or "hinge domain", is used to link the antigen-binding domain to the transmembrane domain. In some instances, a "stalk domain" or "stalk region" comprise any oligonucleotide- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. In some embodiments, it is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition.

In some instances, the stalk region comprises the hinge region from IgGI. In alternative instances, the stalk region comprises the CH2CH3 region of immunoglobulin and optionally portions of CD3. In some cases, the stalk region comprises a CD8a hinge region (SEQ ID NO 29; SEQ ID NO 170), an IgG4-Fc 12 amino acid hinge region (ESKYGPPCPPCP) or IgG4 hinge regions as described in WO/2016/073755.

[0242] In some embodiments, the stalk region comprises at least one of a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of CD8 alpha 2X (SEQ ID NO 30; SEQ ID NO 171), CD8 alpha 3X (SEQ ID NO 31; SEQ ID NO 172) or CD8 alpha 4X (SEQ ID NO 32; SEQ ID NO 173).

[0243] In other embodiments, between the extracellular domain and the transmembrane domain of the CAR, there is incorporated a spacer. A spacer can comprise a stalk region and a stalk extension region. In one embodiment, a spacer can include a single stalk region. In another embodiment, a spacer can comprise a stalk region and stalk extension region(s). For example, a spacer can comprise one (1) stalk region and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 stalk regions. In further embodiments, the stalk region can be linked to stalk extension region via a linker.

[0244] The transmembrane domain can be derived from either a natural or a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Suitable transmembrane domains can include the transmembrane region(s) of alpha, beta or zeta chain of the T-cell receptor; or a transmembrane region from CD28, CD3 epsilon, CD3(, CD45, CD4, CD5, CD8alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. Alternatively the transmembrane domain can be synthetic, and can comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine is found at one or both termini of a synthetic transmembrane domain. Optionally, a short oligonucleotide or polypeptide linker, in some embodiments, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of a CAR. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain or a CD3( transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In other embodiments, the transmembrane domain comprises a CD3( transmembrane domain. In some embodiments, the transmembrane region comprises at least one of a polypeptide having at least 9 0% , 9 1 % , 9 2 %, 9 3 %, 94%, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the amino acid sequence of CD8 alpha transmembrane domain (SEQ ID NO 174). In some embodiments, the transmembrane region comprises at least one of a polypeptide having at least 90%, 91%, 9 2 %,

9 3 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of CD28 transmembrane domain (SEQ ID NO 175).

[0245] The intracellular domain can comprise one or more costimulatory domains. Exemplary costimulatory domains include, but are not limited to, CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP1O, DAP12, OX40 (CD134) or fragment or combination thereof. In some instances, a CAR described herein comprises one or more, or two or more of costimulatory domains selected from CD8, CD27, CD28,4-1BB (CD137), ICOS, DAP1O, DAP12, OX40 (CD134) or fragment or combination thereof. In some instances, a CAR described herein comprises one or more, or two or more of costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, OX40 (CD134) or fragment or combination thereof. In some instances, a CAR described herein comprises one or more, or two or more of costimulatory domains selected from CD8, CD28, 4-1BB (CD137), or fragment or combination thereof. In some instances, a CAR described herein comprises one or more, or two or more of costimulatory domains selected from CD28, 4 iBB (CD137), or fragment or combination thereof In some instances, a CAR described herein comprises costimulatory domains CD28 and 4-1BB (CD137) or their respective fragments thereof. In some instances, a CAR described herein comprises costimulatory domains CD28 and OX40 (CD134) or their respective fragments thereof In some instances, a CAR described herein comprises costimulatory domains CD8 and CD28 or their respective fragments thereof. In some instances, a CAR described herein comprises costimulatory domains CD28 or a fragment thereof. In some instances, a CAR described herein comprises costimulatory domains 4-1BB (CD137) or a fragment thereof. In some instances, a CAR described herein comprises costimulatory domains OX40 (CD134) or a fragment thereof In some instances, a CAR described herein comprises costimulatory domains CD8 or a fragment thereof.

[0246] The intracellular signaling domain, also known as cytoplasmic domain, of the CAR of the present disclosure, is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. In some embodiments, the intracellular domain further comprises a signaling domain for T-cell activation. In some instances, the signaling domain for T-cell activation comprises a domain derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b or CD66d. In some cases, the signaling domain for T-cell activation comprises a domain derived from CD3(.

[0247] In some embodiments, the intracellular signaling domain comprises at least one of a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of CD28 (SEQ ID NO 176), CD3 zeta signaling domain (SEQ ID NO 177), 4-1BB signaling domain (SEQ ID NO 178), DNAX-activation protein 10 (DAP 10) signaling domain (SEQ ID NO 179) or DNAX-activation protein 12 (DAP12) signaling domain (SEQ ID NO 180).

CD19 Specific CARs

[0248] CD19 is a cell surface glycoprotein of the immunoglobulin superfamily. In some instances, CD19 has been detected in solid tumors such as pancreatic cancer, liver cancer, and prostate cancer.

[0249] In some embodiments, the antigen binding moiety of a CAR described herein, is specific to CD19. A CD19-specific CAR, when expressed on the cell surface, may redirect the specificity of T cells to human CD19. In embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising a variable domain light chain (VL) and variable domain heavy chain (VH) of a target antigen specific monoclonal anti-CD19 antibody joined by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv are SJ25Cland/or FMC63. In embodiments, the scFvis humanized. Insome embodiments, the antigen binding moiety may comprise VH and VL that are directionally linked, for example, from N to C terminus, VH-linker-VL or VL-linker-VH.

[0250] In some embodiments, described herein include a CD19-specific CAR, in which the antigen binding domain comprises a scFv that binds CD19. In some instances, the antigen binding domain recognizes an epitope on CD19.

[0251] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some embodiments, described herein include a CD19-specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by JCAR14, JCAR15, JCARO17, or 19-28z CAR (Juno Therapeutics). In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from

CD27, CD28, 4-1BB (CD137), ICOS, DAP1, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0252] In some embodiments, described herein include a CD19-specific CAR-T cell comprises a scFv antigen binding domain, and the antigen binding domain recognizes an epitope on CD19 that is also recognized by JCAR14, JCARO15, JCARO17, or 19-28z CAR (Juno Therapeutics). In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3(transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0253] In some embodiments, a CD19-specific CAR-T cell described herein comprises an anti CD19 antibody described in US20160152723.

[0254] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19 (Kite Pharma, Inc.). In some embodiments, described herein include a CD19-specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP1O, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0255] In some embodiments, described herein include a CD19-specific CAR-T cell comprises a scFv antigen binding domain, and the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP1, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0256] In some embodiments, a CD19-specific CAR-T cell described herein comprises an anti CD19 antibody described in W02015187528 or fragment or derivative thereof.

[0257] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTLO19 (Novartis). In some embodiments, described herein include a CD19-specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTLO19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB

(CD137), ICOS, DAP1, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0258] In some embodiments, described herein include a CD19-specific CAR-T cell comprises a scFv antigen binding domain, and the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTLO19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP1, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0259] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19 (Cellectis). In some embodiments, described herein include a CD19-specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP1, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0260] In some embodiments, described herein include a CD19-specific CAR-T cell comprises a scFv antigen binding domain, and the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0261] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401 (Bellicum). In some embodiments, described herein include a CD19-specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP1,DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0262] In some embodiments, described herein include a CD19-specific CAR-T cell comprises a scFv antigen binding domain, and the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401. In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP1, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0263] In some cases, the antigen binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximabravtansine (ImmunoGen Inc./Sanofi-aventis), MOR208 (Morphosys AG/Xencor Inc.), MEDI-551 (Medimmune), denintuzumabmafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomabpaptox (National Cancer Institute), XmAb 5871 (Amgen/Xencor, Inc.), MDX-1342 (Medarex) or AFM11 (Affimed). In some instances, the CD19-specific CAR further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0264] In some embodiments, described herein include a CD19-specific CAR-T cell, in which the antigen binding domain comprises a F(ab')2, Fab', Fab, Fv, or scFv. In some instances, the antigen binding domain recognizes an epitope on CD19. In some cases, the antigen binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximabravtansine (ImmunoGen Inc./Sanofi-aventis), MOR208 (Morphosys AG/Xencor Inc.), MEDI-551 (Medimmune), denintuzumabmafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomabpaptox (National Cancer Institute), XmAb 5871 (Amgen/Xencor, Inc.), MDX-1342 (Medarex) or AFM11 (Affimed). In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3( transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP1O, DAP12, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0265] In some cases, a CD19-specific CAR-T cell described herein comprise a scFv antigen binding domain, and the antigen binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximabravtansine (ImmunoGen Inc./Sanofi-aventis), MOR208 (Morphosys AG/Xencor Inc.), MEDI-551 (Medimmune), denintuzumabmafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomabpaptox (National Cancer Institute), XmAb 5871 (Amgen/Xencor, Inc.), MDX-1342 (Medarex) or AFM11 (Affimed). In some instances, the CD19-specific CAR-T cell further comprises a transmembrane domain selected from a CD8alpha transmembrane domain or a CD3(transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134) or fragment or combination thereof, and a signaling domain from CD3(.

[0266] In embodiments, a CAR described herein comprises CD19 specific CAR (CD19-CD8a CD28-CD3() having at least 90%,91%,92%,93%, 94%, 95%, 96%,97%, 98%,99% or 100% identity with the amino acid sequence of SEQ ID NO 210. In embodiments, a CAR described herein comprises CD19 specific CAR (CD19-CD8a-CD28-CD3( with signal peptide) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 211.

[0267] In some embodiments, the antigen binding moiety of a CAR described herein is specific to CD19. The CD19-specific CAR, when expressed on the cell surface, redirects the specificity of T cells to human CD19. In embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising a variable domain light chain (VL) and variable domain heavy chain (VH) of a target antigen specific monoclonal anti-CD19 antibody joined by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv is anti-CD19 clone FMC63 scFv with whitlow linker (SEQ ID NO 213). In embodiments, the anti CD19 antibody comprises anti-CD19 monoclonal antibody clone FMC63 variable heavy chain (SEQ ID NO 212). In some embodiments, the antigen binding moiety may comprise VH and VL that are directionally linked, for example, from N to C terminus, VH-linker-VL or VL-linker-VH.

Engineered T Cell Receptor (TCR)

[0268] In some embodiments, the chimeric receptor encoded by a polynucleotide described herein, comprises an engineered T-cell receptor. The T cell receptor (TCR) is composed of two chains (a or 7 6) that pair on the surface of the T cell to form a heterodimeric receptor. In some instances, the a TCR is expressed on most T cells in the body and is known to be involved in the recognition of specific MHC-restricted antigens. Each a and p chain are composed of two domains: a constant domain (C) which anchors the protein to the cell membrane and is associated with invariant subunits of the CD3 signaling apparatus; and a variable domain (V) that confers antigen recognition through six loops, referred to as complementarity determining regions (CDRs). In some instances, each of the V domains comprises three CDRs; e.g., CDR1, CDR2 and CDR3 with CDR3 as the hypervariable region. These CDRs interact with a complex formed between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pepMHC) (e.g., HLA-A, HLA-B, HLA-C, HLA-DPA1,HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, or HLA-DRB1 complex). In some instances, the constant domain further comprises ajoining region that connects the constant domain to the variable domain. In some cases, the beta chain further comprises a short diversity region which makes up part of the joining region.

[0269] In some cases, such TCR are reactive to specific tumor antigen, e.g. NY-ESO, Mage A3, Titin. In other cases, such TCR are reactive to specific neoantigens expressed within a patient's tumor (i.e. patient-specific, somatic, non-synonymous mutations expressed by tumors). In some cases, engineered TCRs can be affinity-enhanced.

[0270] In some embodiments, a TCR is described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. For example, there can be several types of alpha chain variable (Va) regions and several types of beta chain variable (Vj) regions distinguished by their framework, CDR1, CDR2, and CDR3 sequences. As such, a Va type can be referred to in IMGT nomenclature by a unique TRAV number. For example, "TRAV21" defines a TCR Va region having unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence which is partly defined by an amino acid sequence which is preserved from TCR to TCR but which also includes an amino acid sequence which varies from TCR to TCR. Similarly, "TRBV5-1" defines a TCR V region having unique framework and CDR1 and CDR2 sequences, but with only a partly defined CDR3 sequence.

[0271] In some cases, the beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD.

[0272] In some instances, the unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the IMGT public database and in "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.

[0273] In some embodiments, an a heterodimeric TCR is, for example, transfected as full length chains having both cytoplasmic and transmembrane domains. In some cases, the TCRs contain an introduced disulfide bond between residues of the respective constant domains, as described, for example, in WO 2006/000830.

[0274] In some instances, TCRs described herein are in single chain format, for example see WO 2004/033685. Single chain formats include up TCR polypeptides of the Va-L-VO, VO-L-Va, Va-Ca--L-VV-,-L-V-CVa-CL-VP-Cp types, wherein Va and VP are TCR a and 0 variable regions respectively, Ca and Cp are TCR a and constant regions respectively, and L is a linker sequence. In certain embodiments single chain TCRs of the present disclosure may have an introduced disulfide bond between residues of the respective constant domains, as described in WO 2004/033685.

[0275] The TCR described herein may be associated with a detectable label, a therapeutic agent or a PK modifying moiety.

[0276] Exemplary detectable labels for diagnostic purposes include, but are not limited to, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.

Additional Genetic Elements

[0277] Although cellular therapies hold great promise for the treatment of human disease, significant toxicities from the cells themselves or from their transgene products have hampered clinical investigation. In embodiments described herein, immune effector cells comprising a CAR or TCR described herein that have been infused into a mammalian subject, e.g., a human, can be ablated in order to regulate the effect of such immune effector cells should toxicity arise from their use. Therefore, certain in embodiments, in addition to the specific chimeric antigen receptor described herein, a second gene is also introduced into an engineered immune effector cell described herein. The second gene is effectively a "kill switch" or "cell tag" that allows for the depletion of CAR or TCR or antigen binding polypeptide containing cells. In certain embodiments, the "kill switch" is a truncated HERI peptide (herein designated HERIt or EGFRt) which comprises at least an antibody binding epitope of HERI or functional fragment thereof, and optionally a signal polypeptide sequence or fragment thereof

[0278] In certain embodiments, the second gene is a HERI tag which is Epidermal Growth Factor Receptor (HERI) or a fragment or variant thereof. In embodiments, the second gene is a HERI tag which is truncated human Epidermal Growth Factor Receptor 1 (for instance HERIt) (SEQ ID NO 76; SEQ ID NO 189). In some cases, the second gene is a variant of a truncated human Epidermal Growth Factor Receptor 1. In some cases, the variant of a truncated HERI is HERt (SEQ ID NO 77; SEQ ID NO 190), HERlt2 (SEQ ID NO 78; SEQ ID NO 191), HERlt3 (SEQ ID NO 79; SEQ ID NO 192), HER1t4 (SEQ ID NO 80; SEQ ID NO 193), HERlt5 (SEQ ID NO 81; SEQ ID NO 194), HERlt6 (SEQ ID NO 82; SEQ ID NO 195), HERlt7 (SEQ ID NO 83; SEQ ID NO 196), HERlt8 (SEQ ID NO 84; SEQ ID NO 197), HERlt9 (SEQ ID NO 85; SEQ ID NO 198), HERlO (SEQ ID NO 86; SEQ ID NO 199) or HERlt1(SEQ ID NO 87; SEQ ID NO 200). In embodiments, at least one of HERI, HERIt, HERtl, HERlt2, HERt3, HER1t4, HERIt5, HERlt6, HERlt7, HERlt8, HERlt9, HERltO and HERt11 provide a safety mechanism by allowing for depletion of infused CAR-T cells through administering FDA approved cetuximab or any antibody that recognizes HERI, HERIt, HERtl, HERlt2, HERt3, HER1t4, HERIt5, HERlt6, HERlt7, HERlt8, HERlt9, HERltO and/or HERIt11.

[0279] In embodiments, the HERIt gene comprises a nucleotide sequence having at least 90%, 91%,92%,93%,94%,95%,96%,97%, 98%,99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 76. In embodiments, the HERt gene comprises a nucleotide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the nucleic acid sequence of SEQ ID NO: 77. In embodiments, the HERlt2 gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6 %, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 78. In embodiments, the HERlt3 gene comprises a nucleotide sequence having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 79. In embodiments, the HER1t4 gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 80. In embodiments, the HERlt5 gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 81. In embodiments, the HERlt6 gene comprises a nucleotide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 9 5%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the nucleic acid sequence of SEQ ID NO: 82. In embodiments, the HERlt7 gene comprises a nucleotide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 9 5%, 9 6 9 7 %, %, 9 8 %, 9 9% or 100% identity with the nucleic acid sequence of SEQ ID NO: 83. In embodiments, the HERlt8 gene comprises a nucleotide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %,

95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 84. In embodiments, the HERlt9 gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 85. In embodiments, the HERltO gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 86. In embodiments, the HERt11 gene comprises a nucleotide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 9 5%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the nucleic acid sequence of SEQ ID NO: 87.

[0280] The truncated HERI sequence, for instance HERIt, HERtl, HERt2, HERlt3, HER1t4, HERIt5, HERlt6, HERlt7, HERlt8, HERlt9, HERlO and/or HERlI eliminates the potential for EGF ligand binding, homo- and hetero- dimerization of EGFR, and EGFR mediated signaling while keeping cetuximab binding to the receptor intact (Ferguson, K., 2008. A structure-based view of Epidermal Growth Factor Receptor regulation. Annu Rev Biophys, Volume 37, pp. 353-373).

[0281] In further embodiments, in addition to the therapeutic target specific chimeric antigen receptor of the present disclosure the second gene introduced is a HERI tag. In some cases, the HERI tag is a full-length HERI polypeptide, or a truncated HERI polypeptide (HERlt1), HERt2, HERt3, HER1t4, HERIt, HERlt6, HERlt7, HERlt8, HERlt9, HERlO or HERt11. In some cases, the HERI tag is a truncated HERI variant. In some cases, the HER tag, for instance HER,HERt, HERt2, HERt3, HER1t4, HERIt, HERlt6, HERlt7, HERlt8, HERlt9, HERl0 or HERlt1 also provides a safety mechanism by allowing for depletion of infused CAR-T cells through administering FDA-approved rituximab therapy. In certain embodiments, the HERI tag has a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 189. In certain embodiments, the HERI tag is a HERt tag and has a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 190. In certain embodiments, the HERI tag is a HERt2 tag and has a polypeptide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or

100% identity with the sequence of SEQ ID NO: 191. In certain embodiments, the HERI tag is a HERlt3 tag and has a polypeptide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 192. In certain embodiments, the HERI tag is a HER1t4 tag and has a polypeptide sequence having at least 9 0% , 9 1 % , 9 2 %, 9 3 %, 9 4 %, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the sequence of SEQ ID NO: 193. In certain embodiments, the HERI tag is a HERlt5 tag and has a polypeptide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the sequence of SEQ ID NO: 194. In certain embodiments, the HERI tag is a HERlt6 tag and has a polypeptide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 195. In certain embodiments, the HERI tag is a HERlt7 tag and has a polypeptide sequence having at least 9 0% , 9 1 % , 9 2 %, 9 3 %, 9 4 %, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the sequence of SEQ ID NO: 196. In certain embodiments, the HERI tag is a HERlt8 tag and has a polypeptide sequence having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the sequence of SEQ ID NO: 197. In certain embodiments, the HERI tag is a HERlt9 tag and has a polypeptide sequence having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 198. In certain embodiments, the HERI tag is a HERlO tag and has a polypeptide sequence having at least 9 0% , 9 1 % , 9 2 %, 93%, 94%, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the sequence of SEQ ID NO: 199. In certain embodiments, the HERI tag is a HERltI1 tag and has a polypeptide sequence having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 9 6 %, 97%, 9 8 %, 9 9 % or 100% identity with the sequence of SEQ ID NO: 200.

[0282] In certain embodiments, the second gene is a CD20 tag (SEQ ID NO 88; SEQ ID NO 201) which is an activated glycosylated phosphoprotein or a fragment or variant thereof. Insome cases, the second gene is a variant of a truncated CD20, CD20tl (SEQ ID NO 89; SEQ ID NO 202). In embodiments, the CD20 tag, the variant of CD20 tag (CD20tl) or the fragment of the CD20 or CD20tl tag provides a safety mechanism by allowing for depletion of infused CAR-T cells through administering an antibody that recognizes CD20. In embodiments, the gene encoding the CD20 tag comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 88. In embodiments, the gene encoding the CD20tl tag comprises a nucleotide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 9 5 %, 9 6 %, 9 7 %, 9 8 %, 9 9% or 100% identity with the nucleic acid sequence of SEQ ID NO: 89.

[0283] In further embodiments, in addition to the therapeutic target specific chimeric antigen receptor of the present disclosure the second gene introduced is a CD20 tag. In some cases, the CD20 tag is a full-length CD20 polypeptide or a truncated CD20 polypeptide (CD20tl).

[0284] In embodiments, a CAR vector comprising a CAR described herein further comprises a full length CD20 tag comprising a nucleic acid sequence having at least 90%, 91%, 9 2 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 88. In embodiments, a CAR vector comprising a CAR described herein further comprises a variant of CD20 tag (CD20tl) comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleic acid sequence of SEQ ID NO: 89.

[0285] In embodiments, the gene encoding the kill tag, for instance the HERlt, HERtl, HERlt2, HERlt3, HER1t4, HERlt5, HERlt6, HERlt7, HERlt8, HERlt9, HERlO or HERltI1 is genetically fused to the CAR or TCR or cytokine via in-frame with a self-cleaving peptide, for example but not restricted to Thosea asignavirus (T2A) peptide. In embodiments, the T2A peptide has an amino acid sequence having at least 90%, 91%, 9 2 %, 9 4 %, 9 5 %, 9 6 %, 9 7 %, 93%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 153. In other embodiments, the gene encoding the kill tag, for instance the HERt, HERtl, HERlt2, HERlt3, HER1t4, HERIt5, HERlt6, HERlt7, HERlt8, HERlt9, HERl or HERt11 is genetically fused to a cytokine at 3' end via in-frame with a self-cleaving peptide, for example but not restricted to Thosea asignavirus (T2A) peptide.

[0286] In embodiments, both genes are cloned into a plasmid. In other embodiments, the cell tag is cloned into a separate lentiviral vector. In other embodiments, the cell tag gene is cloned into the Sleeping Beauty transposon vector backbone in frame with the CAR gene. In yet other embodiments, the cell tag such as HERt, HERtl, HERlt2, HERt3, HER1t4, HERIt5, HERlt6, HERlt7, HERlt8, HERlt9, HERl or HERlt1 is cloned into multiple Sleeping Beauty transposon vectors.

[0287] In certain embodiments, the cell tags have a signal peptide, for instance, GM-CSFRa signal peptide wherein the GM-CSFRa signal peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 163. In certain embodiments, the signal peptide is IgK having a sequence at least 90%, 91%, 9 2 %,

93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 164. In certain embodiments, the signal peptide is IgE having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 165. In certain embodiments, the signal peptide is CD8a having a sequence at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the amino acid sequence of SEQ ID NO: 166. In certain embodiments, the signal peptide is TVB2 (T21A) having a sequence at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 9 6 %, 9 7 %, 9 8 %, 95%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 167. In certain embodiments, the signal peptide is CD52 having a sequence at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %,

95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 168. In certain embodiments, the signal peptide is low-affinity nerve growth factor receptor (LNGFR, TNFRSF16) having a sequence at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 95%, 9 6 %, 9 7 %, 9 8 %, 99

% or 100% identity with the amino acid sequence of SEQ ID NO: 169.In some cases the signal peptide can be selected from GM-CSFRa, IgK, IgE, CD8a, T21A, CD52, low-affinity nerve growth factor receptor variants and fragments thereof.

[0288] In some embodiments, the cell tags have a signal peptide, for instance, GM-CSFRa signal peptide wherein the GM-CSFRa signal peptide is encoded from a nucleotide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %, 95%, 9 6 %, 9 7 %, 9 8 %, 9 9% or 100% identity with the nucleotide acid sequence of SEQ ID NO 20 or SEQ ID NO 21. In some embodiments, the cell tags have a signal peptide, for instance, IgK signal peptide wherein the IgK signal peptide is encoded from a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide acid sequence of SEQ ID NO 22. In some embodiments, the cell tags have a signal peptide, for instance, IgE signal peptide wherein the IgK signal peptide is encoded from a nucleotide sequence having at least 90%, 91%, 9 2 %, 9 3 %, 9 4 %,

95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide acid sequence of SEQ ID NO 23.In some embodiments, the cell tags have a signal peptide, for instance, CD8a signal peptide wherein the CD8a signal peptide is encoded from a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide acid sequence of SEQ ID NO 24 or SEQ ID NO 25. In some embodiments, the cell tags have a signal peptide, for instance, TVB2 (T21A) signal peptide wherein the TVB2 (T21A) signal peptide is encoded from a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide acid sequence of SEQ ID NO 26. In some embodiments, the cell tags have a signal peptide, for instance, CD52 signal peptide wherein the CD52 signal peptide is encoded from a nucleotide sequence having at least 90%, 91%, 9 2 %,

93%, 9 4 %, 9 5%, 9 6 %, 9 7 %, 9 8 %, 9 9 % or 100% identity with the nucleotide acid sequence of

SEQ ID NO 27. In some embodiments, the cell tags have a signal peptide, for instance, low affinity nerve growth factor receptor (LNGFR, TNFRSF16) signal peptide wherein the low affinity nerve growth factor receptor (LNGFR, TNFRSF16) signal peptide is encoded from a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide acid sequence of SEQ ID NO 28.

[0289] A signal peptide is a sequence of amino acids typically located at the N-terminus of a newly synthesized protein or polypeptide which directs the protein or polypeptide to the cell surface. In some embodiments, the signal peptide directs the polypeptide to the cell surface to be inserted (e.g., via a transmembrane domain) into the cellular membrane. In some embodiments, a polypeptide construct described herein is synthesized with the signal peptide, but then post translationally processed to cleave the signal peptide such that the mature polypeptide construct lacks the signal peptide amino acid sequence. In other embodiments, the signal peptide sequence is not cleaved and remains in the mature polypeptide construct.

[0290] The present disclosure provides for a polypeptide construct comprising any known or unknown signal peptide capable of directing and/or trafficking the polypeptide construct to the cell surface. For example, in some embodiments, a polypeptide construct comprises a signal sequence corresponding to the signal peptide of GMCSFRa, Ig Kappa, Immunoglobulin E, CD8a, TVB2 (T21A), CD52 or Low-affinity nerve growth factor receptor (LNGFR, TNFRSF16).

[0291] In embodiments, the signal peptide is encoded by a polynucleotide comprising a nucleotide sequence which has at least 70%, 75%, 8 0%, 8 5 %, 90%, 95%, 99%, or 100% identity with a nucleotide sequence selected from the list consisting of SEQ ID NO: 20-28. In embodiments, the signal peptide comprises an amino acid sequence which has at least 70%, 75%, 80% , 8 5 %, 90%, 95%, 99%, or 100% identity with an amino acid sequence selected from the list consisting of SEQ ID NO: 163-169.

Modified Effector Cells

[0292] Provided are effector cells (also referred to as immune effector cells) modified to express one or more heterologous genes or genes regulated by gene-switch polypeptides disclosed herein.

[0293] "T cell" or "T lymphocyte" as used herein is a type of lymphocyte that plays a central role in cell-mediated immunity. They may be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface.

[0294] In some embodiments, modified effector cells are modified immune cells that comprise T cells and/or natural killer cells. T cells or T lymphocytes are a subtype of white blood cells that are involved in cell-mediated immunity. Exemplary T cells include T helper cells, cytotoxic T cells, TH17 cells, stem memory T cells (TSCM), naive T cells, memory T cells, effector T cells, regulatory T cells, or natural killer T cells.

[0295] "T helper cells" (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. In some instances, TH cells are known as CD4+ T cells due to expression of the CD4 glycoprotein on the cell surfaces. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including THI, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate different types of immune responses. Signaling from the APC directs T cells into particular subtypes.

[0296] "Cytotoxic T cells" (TC cells or CTLs) destroy virus-infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein on their surfaces. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0297] "Memory T cells" are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise subtypes: stem memory T cells (TSCM), central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells may express the cell surface proteins CD45RO, CD45RA and/or CCR7.

[0298] "Regulatory T cells" (Treg cells), formerly known as suppressor T cells, play a role in the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress autoreactive T cells that escaped the process of negative selection in the thymus.

[0299] "Natural killer T cells" (NKT cells) bridge the adoptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CDld. Once activated, these cells can perform functions ascribed to both Th and Tc cells (i.e., cytokine production and release of cytolytic/cell killing molecules). They are also able to recognize and eliminate some tumor cells and cells infected with herpes viruses. Natural killer (NK) cells are a type of cytotoxic lymphocyte of the innate immune system. In some instances, NK cells provide a first line defense against viral infections and/or tumor formation. NK cells can detect MHC presented on infected or cancerous cells, triggering cytokine release, and subsequently induce lysis and apoptosis. NK cells can further detect stressed cells in the absence of antibodies and/or MHC, thereby allowing a rapid immune response.

Modified Effector Cell Doses

[0300] In some embodiments, an amount of modified effector cells is administered to a subject in need thereof and the amount is determined based on the efficacy and the potential of inducing a cytokine-associated toxicity. In some cases, an amount of modified effector cells comprises about 105 to about 109 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 10 5 to about 10 8 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 10 5 to about 10 7 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 106 to about 10 9 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 106 to about 10 8 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 10 7 to about 10 9 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 105 to about 106 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 106 to about 10 7 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 10 7 to about 10 8 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 10 8 to about 10 9 modified effector cells/kg. In some instances, an amount of modified effector cells comprises about 10 9 modified effector cells/kg. In some instances, an amount of modified effector cells comprises about 10 8 modified effector cells/kg. In some instances, an amount of modified effector cells comprises about 10 7 modified effector cells/kg. In some instances, an amount of modified effector cells comprises about 106 modified effector cells/kg. In some instances, an amount of modified effector cells comprises about 10 5 modified effector cells/kg.

[0301] In some embodiments, the modified effector cells are modified T cells. In some instances, the modified T cells are CAR-T cells. In some cases, an amount of CAR-T cells comprises about 105 to about 10 9 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 105 to about 10 8 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 10' to about 10 7 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 106 to about 10 9 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 106 to about 10 8 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 10 7 to about 10 9 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 105 to about 106 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 106 to about 10 7 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 10 7 to about 10 8 CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about 10 8 to about 10 9 CAR-T cells/kg. In some instances, an amount of CAR-T cells comprises about 109 CAR-T cells/kg. In some instances, an amount of CAR-T cells comprises about 10 8 CAR-T cells/kg. In some instances, an amount of CAR-T cells comprises about 10 7 CAR-T cells/kg. In some instances, an amount of CAR-T cells comprises about 106 CAR-T cells/kg. In some instances, an amount of CAR-T cells comprises about 105 CAR-T cells/kg.

[0302] In some embodiments, the CAR-T cells are CD19-specific CAR-T cells. In some cases, an amount of CD19-specific CAR-T cells comprises about 105 to about 10 9 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 10 5 to about 108 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 10 5 to about 10 7 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 106 to about 109 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 106 to about 108 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 107 to about 109 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 10 5 to about 106 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 106 to about 10 7 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 107 to about 108 CAR-T cells/kg. In some cases, an amount of CD19-specific CAR-T cells comprises about 108 to about 109 CAR-T cells/kg. In some instances, an amount of CD19-specific CAR-T cells comprises about 109 CAR-T cells/kg. In some instances, an amount of CD19-specific CAR-T cells comprises about 108 CAR-T cells/kg. In some instances, an amount of CD19-specific CAR-T cells comprises about 10 7 CAR-T cells/kg. In some instances, an amount of CD19-specific CAR T cells comprises about 106 CAR-T cells/kg. In some instances, an amount of CD19-specific CAR-T cells comprises about 10 5 CAR-T cells/kg.

[0303] In some embodiments, the modified T cells are engineered TCR T-cells. In some cases, an amount of engineered TCR T- cells comprises about 10 5 to about 10 9 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 10 5 to about 108 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 10 5 to about 107 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 106 to about 10 9 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 106 to about 10 8 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 10 7 to about 109 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 105 to about 106 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 106 to about 107 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 107 to about 108 TCR cells/kg. In some cases, an amount of engineered TCR cells comprises about 108 to about 109 TCR cells/kg. In some instances, an amount of engineered TCR cells comprises about 10 9 TCR cells/kg. In some instances, an amount of engineered TCR cells comprises about 108 TCR cells/kg. In some instances, an amount of engineered TCR cells comprises about 107 TCR cells/kg. In some instances, an amount of engineered TCR cells comprises about 106 TCR cells/kg. In some instances, an amount of engineered TCR cells comprises about 105 TCR cells/kg.

Indications

[0304] In some embodiments, disclosed herein are methods of administering a modified effector cell encoding a polynucleotide described herein to a subject having a disorder, for instance a cancer or an infectious disease. In some cases, the cancer is a cancer associated with an expression of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-Al, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 or VEGF-R2.

[0305] In some embodiments, disclosed herein are methods of administering a polynucleotide, polypeptide or a modified effector cell encoding a polynucleotide described herein, to a subject having a cancer associated with an overexpression of CD19. In some embodiments, disclosed herein are methods of administering a modified effector cell to a subject having a cancer associated with an overexpression of CD33. In some embodiments, disclosed herein are methods of administering a modified effector cell to a subject having a cancer associated with an overexpression of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-Al, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 or VEGF-R2. In some cases, the cancer is a metastatic cancer. In other cases, the cancer is a relapsed or refractory cancer.

[0306] In some cases, a cancer is a solid tumor or a hematologic malignancy. In some instances, the cancer is a solid tumor. In other instances, the cancer is a hematologic malignancy.

In some cases, the cancer is a metastatic cancer. In some cases, the cancer is a relapsed or refractory cancer.

[0307] In some instances, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, anal cancer; appendix cancer; bile duct cancer (i.e., cholangiocarcinoma); bladder cancer; brain tumor; breast cancer; cervical cancer; colon cancer; cancer of Unknown Primary (CUP); esophageal cancer; eye cancer; fallopian tube cancer; gastroenterological cancer; kidney cancer; liver cancer; lung cancer; medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreatic cancer; parathyroid disease; penile cancer; pituitary tumor; prostate cancer; rectal cancer; skin cancer; stomach cancer; testicular cancer; throat cancer; thyroid cancer; uterine cancer; vaginal cancer; vulvar cancer; or glioblastoma.

[0308] "Glioblastoma" or "glioblastoma multiforme" (GBM) is an aggressive neuroepithelial brain cancer. GBM may originate from glial type cells, astrocytes, oligodendrocyte progenitor cells, or neural stem cells. Four subtypes of glioblastoma have been identified. The classical subtype, a majority of GBM, carries extra copies of the epidermalgrowthfactorreceptor (EGFR) gene, and most have higher than normal expression of epidermal growth factor receptor (EGFR). In a subset of the cases, EGFR amplification is accompanied by gene rearrangement, the most common of which is EGFR variant III (EGFRvIII). The gene TP53 (p53), which is often mutated in glioblastoma, is rarely mutated in the classical subtype. The proneural subtype often has high rates of alterations in TP53 (p53), and in PDGFRA, the gene encoding platelet derived growth factor receptor A, and in IDH, the gene encoding isocitrate dehydrogenase-1. The Mesenchymal subtype is characterized by high rates of mutations or other alterations in NF1, the gene encoding neurofibromin 1 and fewer alterations in the EGFR gene and less expression of EGFR than other types. The Neural subtype was typified by the expression of neuron markers such as NEFL, GABRA, SYT1 and SLC12A5. Other genetic alterations have been described in glioblastoma, and the majority of them are clustered in two pathways, the RB and the PI3K/AKT. Glioblastomas have alterations in 68-78% and 88% of these pathways, respectively.

[0309] In some instances, the cancer is a hematologic malignancy. In some cases, a hematologic malignancy comprises a lymphoma, a leukemia, a myeloma, or a B-cell malignancy. In some cases, a hematologic malignancy comprises a lymphoma, a leukemia or a myeloma. In some instances, exemplary hematologic malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some embodiments, the hematologic malignancy comprises a myeloid leukemia. In some embodiments, the hematologic malignancy comprises acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0310] In some instances, disclosed herein are methods of administering to a subject having a hematologic malignancy selected from chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis a modified effector cell described herein. In some instances, disclosed herein are methods of administering to a subject having a hematologic malignancy selected from AML or CIL a modified effector cell to the subject.

[0311] In other cases, disclosed herein are methods of administering to a subject having an infection due to an infectious disease. An infectious disease can be a disease resulting from a bacterial, viral or fungi infection. In other instances, exemplary viral pathogens include those of the families of Adenoviridae, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), JC virus, BK virus, HSV, HHV family of viruses, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys . Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella.

Viral Based Delivery System

[0312] The present disclosure also provides delivery systems, such as viral-based systems, in which a nucleic acid described herein is inserted. Representative viral expression vectors include, but are not limited to, adeno-associated viral vectors, adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), lentivirus-based vectors (e.g., the lentiviral-based pLPI from Life Technologies (Carlsbad, Calif)), retroviral vectors (e.g., the pFB-ERV plus pCFB-EGSH), and herpes virus-based vectors. In an embodiment, the viral vector is a lentivirus vector. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In an additional embodiment, the viral vector is an adeno-associated viral vector. In a further embodiment, the viral vector is a retroviral vector. In general, and in embodiments, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

[0313] Additional suitable vectors include integrating expression vectors, which may randomly integrate into the host cell's DNA, or may include a recombination site to enable the specific recombination between the expression vector and the host cell's chromosome. Such integrating expression vectors may utilize the endogenous expression control sequences of the host cell's chromosomes to effect expression of the desired protein. Examples of vectors that integrate in a site specific manner include, for example, components of the flp-in system from Invitrogen (Carlsbad, Calif.) (e.g., pcDNATM5/FRT), or the cre-lox system, such as can be found in the pExchange-6 Core Vectors from Stratagene (La Jolla, Calif). Examples of vectors that randomly integrate into host cell chromosomes include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Invitrogen (Carlsbad, Calif), and pCI or pFN10A (ACT) FLEXITM from Promega (Madison, Wis.). Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0314] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.

[0315] Another example of a suitable promoter is human elongation growth factor 1 alpha 1 (hEFlal). In embodiments, the vector construct comprising the CARs and/or TCRs of the present disclosure comprises hEFlal functional variants.

[0316] However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Other examples of inducible promoters include, but are not limited to tissue specific promoters as described herein. In some embodiments, the promoter comprises NF-xB binding site (SEQ ID NOS 44-46), nuclear factor of activated T cells (NFAT) response element (SEQ ID NO 51), 6 site GAL4-inducible proximal factor binding element (PFB) (SEQ ID NO 62) or synthetic 5' UTR based on RPL6 (SEQ ID NO 64). In certain embodiments, the promoter can be any one or more of: IL-2 core promoter, IL-2 minimal promoter, IL-2 enhancer and promoter variant, (NF KB)1-IL2 promoter variant, (NF-x]B) 3 -IL2 promoter variant, (NF-KB)-IL2 promoter variant, IX NFAT response elements-IL2 promoter variant, 3X NFAT response elements-IL2 promoter variant, 6X NFAT response elements-IL2 promoter variant, human EEF1A1 promoter variant, human EEF1A1 promoter and enhancer, human UBC promoter and synthetic minimal promoter 1. In certain embodiments, the promoter nucleotides comprise disclosed in Table 2.

[0317] In order to assess the expression of a CAR or TCR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neomycin resistance gene (neo) and ampicillin resistance gene and the like. In some embodiments, a truncated epidermal growth factor receptor (HERIt) tag may be used as a selectable marker gene.

[0318] Reporter genes can be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0319] In some embodiments, the vectors comprise a hEFlal promoter to drive expression of transgenes, a bovine growth hormone polyA sequence to enhance transcription, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), as well as LTR sequences derived from the pFUGW plasmid.

[0320] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0321] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). In embodiments, a method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection or polyethylenimine (PEI) Transfection.

[0322] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0323] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0324] In the case where a viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0325] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20o C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

Non-Viral Based Delivery System

[0326] In some instances, polynucleotides encoding gene-switch polypeptides for expressing CARs and/or TCRs described herein can also be introduced into T cells using non-viral based delivery systems, such as the "Sleeping Beauty (SB) Transposon System," which refers a synthetic DNA transposon system for introducing DNA sequences into the chromosomes of vertebrates. Some exemplary embodiments of the system are described, for example, in U.S. Pat. Nos. 6,489,458, 8,227,432, 9,228,180 and W02016/145146. The Sleeping Beauty transposon system is composed of a Sleeping Beauty (SB) transposase and a SB transposon. In embodiments, the Sleeping Beauty transposon system can include the SB11 transposon system, the SB100X transposon system, or the SB110 transposon system and any other variants thereof.

[0327] DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner. Transposition is a precise process in which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome. As do other Tcl/mariner-type transposases, SB transposase inserts a transposon into a TA dinucleotide base pair in a recipient DNA sequence. The insertion site can be elsewhere in the same DNA molecule, or in another DNA molecule (or chromosome). In mammalian genomes, including humans, there are approximately 200 million TA sites. The TA insertion site is duplicated in the process of transposon integration. This duplication of the TA sequence is a hallmark of transposition and used to ascertain the mechanism in some experiments. The transposase can be encoded either within the transposon or the transposase can be supplied by another source, for instance a DNA or mRNA source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons are most useful as genetic tools because after insertion they cannot independently continue to excise and re-insert. SB transposons envisaged to be used as non-viral vectors for introduction of genes into genomes of vertebrate animals and for gene therapy. Briefly, the Sleeping Beauty (SB) system (Hackett et al., Mol Ther

18:674-83, (2010)) was adapted to genetically modify the T cells (Cooper et al., Blood 105:1622 31, (2005)). This involved two steps: (i) the electro-transfer of DNA plasmids expressing a SB transposon [i.e., chimeric antigen receptor (CAR) to redirect T-cell specificity (Jin et al., Gene Ther 18:849-56, (2011); Kebriaei et al., Hum Gene Ther 23:444-50, (2012)) and SB transposase and (ii) the propagation and expansion of T cells stably expressing integrants on designer artificial antigen-presenting cells (AaPC)derived from the K562 cell line (also known as AaPCs (Activating and Propagating Cells). In one embodiment, the SB transposon system includes coding sequence encoding tdIL-15, an IL-21 and/or a chimeric antigen receptor. Such systems are described for example in Singh et al., Cancer Res (8):68 (2008). April 15, 2008 and Maiti et al., J Immunother. 36(2): 112-123 (2013), incorporated herein by reference in their entireties.

[0328] In some embodiments, a polynucleotide encoding a CAR or a TCR, one or more gene switch polypeptides and mbIL-15 is encoded in one or more Sleeping Beauty transposon(s), and the SB transposase is encoded in a separate vector. In embodiments, the CD19 specific CAR is encoded in a transposon DNA plasmid vector, mb-IL15 is encoded in a second transposon DNA plasmid vector, and the SB transposase is encoded in a third DNA plasmid vector. In some embodiments, the mbIL-15 is encoded with a cell tag. Examples of cell tags can include truncated epidermal growth factor receptor tag (HERt), HERtl, HERlt2, HERt3, HERlt4, HERIt5, HERlt6, HERlt7, HERlt8, HERlt9, HERl, HERl1, CD20 and CD20tl. CD20 tag or any other appropriate cell tags for use as a depletion or kill switch, or enrichment marker. Non-limiting exemplary gene switch vector system comprising a cell tag is illustrated in FIG. 2A-2D, FIG. 3,FIG. 19A-19B, FIG. 20A, FIG. 22, FIG. 24A-24D, and FIG. 25A-25B.

[0329] In some embodiments, HERIt provides a safety mechanism by allowing for depletion of infused CAR-T cells through administering FDA approved cetuximab or any antibody that recognizes HERIt. In some embodiments, CD20 also provides a safety mechanism by allowing for depletion of infused CAR-T cells through administering FDA-approved rituximab therapy.

[0330] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, molecular assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the present disclosure. In embodiments, a modified effector cell described herein and other genetic elements are delivered to a cell using the SB11 transposon system, the SB1OOX transposon system, the SB110 transposon system, the piggyBac transposon system (see, e.g., Wilson et al, "PiggyBac Transposon-mediated Gene Transfer in Human Cells," Molecular Therapy 15:139-145 (2007), incorporated herein by reference in its entirety) and/or the piggyBac transposon system (see, e.g., Mitra et al., "Functional characterization of piggyBac from the bat Myotis lucifugus unveils an active mammalian DNA transposon," Proc. Natl. Acad. Sci USA 110:234-239 (2013). Additional transposases and transposon systems are provided in U.S. Patent Nos.; 6,489,458; 6,613,752, 7,148,203; 7,985,739; 8,227,432; 9,228,180; U.S. Patent Publn. No. 2011/0117072; Mates et al., Nat Genet, 41(6):753-61 (2009). doi: 10.1038/ng.343. Epub 2009 May 3, Gene Ther., 18(9):849-56 (2011). doi: 10.1038/gt.2011.40. Epub 2011 Mar 31 and in Ivics et al., Cell. 91(4):501-10, (1997), each of which is incorporated herein by reference in their entirety.

[0331] Additional suitable non-viral systems can include integrating expression vectors, which may randomly integrate into the host cell's DNA, or may include a recombination site to enable the specific recombination between the expression vector and the host cell's chromosome. Targeted integration of transgenes into predefined genetic loci is a desirable goal for many applications. First, a first recombination site for a site-specific recombinase is inserted at a genomic site, either at a random or at a predetermined location. Subsequently, the cells are transfected with a plasmid carrying the gene or DNA of interest and the second recombination site and a source for recombinase (expression plasmid, RNA, protein, or virus-expressing recombinase). Recombination between the first and second recombination sites leads to integration of plasmid DNA.

[0332] Such integrating expression vectors may utilize the endogenous expression control sequences of the host cell's chromosomes to effect expression of the desired protein. In some embodiments, targeted integration is promoted by the presence of sequences on the donor polynucleotide that are homologous to sequences flanking the integration site. For example, targeted integration using the donor polynucleotides described herein may be achieved following conventional transfection techniques, e.g. techniques used to create gene knockouts or knockins by homologous recombination. In other embodiments, targeted integration is promoted both by the presence of sequences on the donor polynucleotide that are homologous to sequences flanking the integration site, and by contacting the cells with donor polynucleotide in the presence of a site-specific recombinase. By a site-specific recombinase, or simply a recombinase, it is meant is a polypeptide that catalyzes conservative site-specific recombination between its compatible recombination sites. As used herein, a site-specific recombinase includes native polypeptides as well as derivatives, variants and/or fragments that retain activity, and native polynucleotides, derivatives, variants, and/or fragments that encode a recombinase that retains activity.

[0333] Also provided herein is a system for integrating heterologous genes in a host cell, said system comprising one or more gene expression cassettes. In some instances, the system includes a first gene expression cassette comprising a first polynucleotide encoding a first polypeptide construct. In other instances, the system can include a second gene expression cassette comprising a second polynucleotide encoding a second polypeptide construct. In yet other instances, the system can include a third gene expression cassette. In one embodiment, one of the gene expression cassettes can comprise a gene switch polynucleotide encoding one or more of: (i) a transactivation domain; (ii) nuclear receptor ligand binding domain; (iii) a DNA binding domain; and (iv) ecdysone receptor binding domain. In another embodiment, the system further includes recombinant attachment sites; and a serine recombinase; such that upon contacting said host cell with at least said first gene expression cassette, in the presence of said serine recombinase, said heterologous genes are integrated in said host cell.

[0334] In some instances, the system further comprises a ligand; such that upon contacting said host cell, in the presence of said ligand, said heterologous gene are expressed in said host cell. In one instance, the system also includes recombinant attachment sites. In some instances, one recombination attachment site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB). In one instance, the host cell is an eukaryotic cell. In another instance, the host cell is a human cell. In further instances, the host cell is a T cell or NK cell.

[0335] In one embodiment, the heterologous gene in the system described above comprises a CAR. In some embodiments, the CAR binds at least one of CD19, CD33, BCMA, CD44, a Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MIUC-1, MAGE-Al, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 or VEGF-R2.

[0336] In another embodiment, the system includes a heterologous gene comprising a cytokine. In one instance, the cytokine comprises at least one of IL-15, IL-12, IL-21, and a fusion of L-15

and IL-15Ra. In one embodiment, the system includes a heterologous gene comprising at least one cell tag. In one instance, said cell tag comprises at least one of HERt and CD20. In some embodiments, the mbIL-15 is encoded with a cell tag. Examples of cell tags can include truncated epidermal growth factor receptor tag (HERIt), CD20 tag or any other appropriate cell tags for use as a depletion or kill switch, or enrichment marker. Exemplary sequences of cell tags are as below:

Table 3. Cell tag amino acid sequences and polynucleotide sequences

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO CGCAAAGTGTGTAACGGAA TAGGTATTGGTGAATTTAA AGACTCACTCTCCATAAAT GCTACGAATATTAAACACT TCAAAAACTGCACCTCCAT CAGTGGCGATCTCCACATC CTGCCGGTGGCATTTAGGG GTGACTCCTTCACACATACT CCTCCTCTGGATCCACAGG AACTGGATATTCTGAAAAC CGTAAAGGAAATCACAGGG TTTTTGCTGATTCAGGCTTG GCCTGAAAACAGGACGGAC CTCCATGCCTTTGAGAACCT AGAAATCATACGCGGCAGG RKVCNGIGIGEFKDSLSINA ACCAAGCAACATGGTCAGT TNIKFKNCTSISGDLHILP TTTCTCTTGCAGTCGTCAGC VAFRGDSFTHTPPLDPQEL CTGAACATAACATCCTTGG DILKTVKEITGFLLIQAWPE GATTACGCTCCCTCAAGGA NRTDLHAFENLEIIRGRTK GATAAGTGATGGAGATGTG QHGQFSLAVVSLNITSLGL ATAATTTCAGGAAACAAAA RSLKEISDGDVIISGNKNLC ATTTGTGCTATGCAAATACA YANTINWKKLFGTSGQKT TruncatedEGFR ATAAACTGGAAAAAACTGT KIISNRGENSCKATGQVCH (huEGFRt) 76 TTGGGACCTCCGGTCAGAA 189 ALCSPEGCWGPEPRDCVSC (Herit) AACCAAAATTATAAGCAAC RNVSRGRECVDKCNLLEG AGAGGTGAAAACAGCTGCA EPREFVENSECIQCHPECLP AGGCCACAGGCCAGGTCTG QAMNITCTGRGPDNCIQCA CCATGCCTTGTGCTCCCCCG HYIDGPHCVKTCPAGVMG AGGGCTGCTGGGGCCCGGA ENNTLVWKYADAGHVCH GCCCAGGGACTGCGTCTCTT LCHPNCTYGCTGPGLEGCP GCCGGAATGTCAGCCGAGG TNGPKIPSIATGMVGALLL CAGGGAATGCGTGGACAAG LLVVALGIGLFM TGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGA GAACTCTGAGTGCATACAG TGCCACCCAGAGTGCCTGC CTCAGGCCATGAACATCAC CTGCACAGGACGGGGACCA GACAACTGTATCCAGTGTG CCCACTACATTGACGGCCC CCACTGCGTCAAGACCTGC CCGGCAGGAGTCATGGGAG AAAACAACACCCTGGTCTG GAAGTACGCAGACGCCGGC CATGTGTGCCACCTGTGCCA TCCAAACTGCACCTACGGA TGCACTGGGCCAGGTCTTG

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO AAGGCTGTCCAACGAATGG GCCTAAGATCCCGTCCATC GCCACTGGGATGGTGGGGG CCCTCCTCTTGCTGCTGGTG GTGGCCCTGGGGATCGGCC TCTTCATG CGCAAAGTGTGTAACGGAA TAGGTATTGGTGAATTTAA AGACTCACTCTCCATAAAT GCTACGAATATTAAACACT TCAAAAACTGCACCTCCAT CAGTGGCGATCTCCACATC CTGCCGGTGGCATTTAGGG GTGACTCCTTCACACATACT CCTCCTCTGGATCCACAGG AACTGGATATTCTGAAAAC CGTAAAGGAAATCACAGGG TTTTTGCTGATTCAGGCTTG GCCTGAAAACAGGACGGAC CTCCATGCCTTTGAGAACCT RKVCNGIGIGEFKDSLSINA AGAAATCATACGCGGCAGG TNIKHFKNCTSISGDLHILP ACCAAGCAACATGGTCAGT VAFRGDSFTHTPPLDPQEL TTTCTCTTGCAGTCGTCAGC DILKTVKEITGFLLIQAWPE CTGAACATAACATCCTTGG NRTDLHAFENLEIIRGRTK esgRtrnHated GATTACGCTCCCTCAAGGA QHGQFSLAVVSLNITSLGL t ddesigen 77 GATAAGTGATGGAGATGTG 190 RSLKEISDGDVIISGNKNLC trun ctle)sign ATAATTTCAGGAAACAAAA YANTINWKKLFGTSGQKT ATTTGTGCTATGCAAATACA KIISNRGENSCKATGQVCH ATAAACTGGAAAAAACTGT ALCSPEGCWGPEPRDCVSG TTGGGACCTCCGGTCAGAA GGGSGGGGSGGGGSGGGG AACCAAAATTATAAGCAAC SFWVLVVVGGVLACYSLL AGAGGTGAAAACAGCTGCA VTVAFIIFWVRSKRS AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCT GGTGGCGGTGGCTCGGGCG GTGGTGGGTCGGGTGGCGG CGGATCTGGTGGCGGTGGC TCGTTTTGGGTGCTGGTGGT GGTTGGTGGAGTCCTGGCTT GCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCT GGGTGAGGAGTAAGAGGAG C EGFR truncated CGCAAAGTGTGTAACGGAA RKVCNGIGIGEFKDSLSINA design 2 (Herl 78 TAGGTATTGGTGAATTTAA TNIKHFKNCTSISGDLHILP

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO truncated design AGACTCACTCTCCATAAAT VAFRGDSFTHTPPLDPQEL 2) (HERlt2) GCTACGAATATTAAACACT DILKTVKEITGFLLIQAWPE TCAAAAACTGCACCTCCAT NRTDLHAFENLEIIRGRTK CAGTGGCGATCTCCACATC QHGQFSLAVVSLNITSLGL CTGCCGGTGGCATTTAGGG RSLKEISDGDVIISGNKNLC GTGACTCCTTCACACATACT YANTINWKKLFGTSGQKT CCTCCTCTGGATCCACAGG KIISNRGENSCKATGQVCH AACTGGATATTCTGAAAAC ALCSPEGCWGPEPRDCVSC CGTAAAGGAAATCACAGGG RNVSRGRECVDKGGGGSG TTTTTGCTGATTCAGGCTTG GGGSGGGGSGGGGSFWVL GCCTGAAAACAGGACGGAC VVVGGVLACYSLLVTVAFI CTCCATGCCTTTGAGAACCT IFWVRSKRS AGAAATCATACGCGGCAGG ACCAAGCAACATGGTCAGT TTTCTCTTGCAGTCGTCAGC CTGAACATAACATCCTTGG GATTACGCTCCCTCAAGGA GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCTT GCCGGAATGTCAGCCGAGG CAGGGAATGCGTGGACAAG GGTGGCGGTGGCTCGGGCG GTGGTGGGTCGGGTGGCGG CGGATCTGGTGGCGGTGGC TCGTTTTGGGTGCTGGTGGT GGTTGGTGGAGTCCTGGCTT GCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCT GGGTGAGGAGTAAGAGGAG C CGCAAAGTGTGTAACGGAA RKVCNGIGIGEFKDSLSINA TAGGTATTGGTGAATTTAA TNIKHFKNCTSISGDLHILP EGFR truncated AGACTCACTCTCCATAAAT VAFRGDSFTHTPPLDPQEL design 3 (Herl 7 GCTACGAATATTAAACACT 192 DILKTVKEITGFLLIQAWPE truncated design TCAAAAACTGCACCTCCAT NRTDLHAFENLEIIRGRTK 3) (HERlt3) CAGTGGCGATCTCCACATC QHGQFSLAVVSLNITSLGL CTGCCGGTGGCATTTAGGG RSLKEISDGDVIISGNKNLC GTGACTCCTTCACACATACT YANTINWKKLFGTSGQKT

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO CCTCCTCTGGATCCACAGG KIISNRGENSCKATGQVCH AACTGGATATTCTGAAAAC ALCSPEGCWGPEPRDCVSC CGTAAAGGAAATCACAGGG RNVSRGRECVDKCNLLEG TTTTTGCTGATTCAGGCTTG EPREFVENSECIQGGGGSG GCCTGAAAACAGGACGGAC GGGSGGGGSGGGGSFWVL CTCCATGCCTTTGAGAACCT VVVGGVLACYSLLVTVAFI AGAAATCATACGCGGCAGG IFWVRSKRS ACCAAGCAACATGGTCAGT TTTCTCTTGCAGTCGTCAGC CTGAACATAACATCCTTGG GATTACGCTCCCTCAAGGA GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCTT GCCGGAATGTCAGCCGAGG CAGGGAATGCGTGGACAAG TGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGA GAACTCTGAGTGCATACAG GGTGGCGGTGGCTCGGGCG GTGGTGGGTCGGGTGGCGG CGGATCTGGTGGCGGTGGC TCGTTTTGGGTGCTGGTGGT GGTTGGTGGAGTCCTGGCTT GCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCT GGGTGAGGAGTAAGAGGAG C CGCAAAGTGTGTAACGGAA RKVCNGIGIGEFKDSLSINA TAGGTATTGGTGAATTTAA TNIKHFKNCTSISGDLHILP AGACTCACTCTCCATAAAT VAFRGDSFTHTPPLDPQEL EGFR truncated GCTACGAATATTAAACACT DILKTVKEITGFLLIQAWPE design 4 (Herd TCAAAAACTGCACCTCCAT NRTDLHAFENLEIIRGRTK design4(Hin 80 CAGTGGCGATCTCCACATC 193 QHGQFSLAVVSLNITSLGL truc teddesign CTGCCGGTGGCATTTAGGG RSLKEISDGDVIISGNKNLC GTGACTCCTTCACACATACT YANTINWKKLFGTSGQKT CCTCCTCTGGATCCACAGG KIISNRGENSCKATGQVCH AACTGGATATTCTGAAAAC ALCSPEGCWGPEPRDCVSC CGTAAAGGAAATCACAGGG RNVSRGRECVDKCNLLEG

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO TTTTTGCTGATTCAGGCTTG EPREFVENSECIQCHPECLP GCCTGAAAACAGGACGGAC QAMNITCTGRGPDNCIQGG CTCCATGCCTTTGAGAACCT GGSGGGGSGGGGSFWVLV AGAAATCATACGCGGCAGG VVGGVLACYSLLVTVAFII ACCAAGCAACATGGTCAGT FWVRSKRS TTTCTCTTGCAGTCGTCAGC CTGAACATAACATCCTTGG GATTACGCTCCCTCAAGGA GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCTT GCCGGAATGTCAGCCGAGG CAGGGAATGCGTGGACAAG TGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGA GAACTCTGAGTGCATACAG TGCCACCCAGAGTGCCTGC CTCAGGCCATGAACATCAC CTGCACAGGACGGGGACCA GACAACTGTATCCAGGGCG GAGGCGGAAGCGGAGGCG GAGGCTCCGGCGGAGGCGG AAGCTTTTGGGTGCTGGTG GTGGTTGGTGGAGTCCTGG CTTGCTATAGCTTGCTAGTA ACAGTGGCCTTTATTATTTT CTGGGTGAGGAGTAAGAGG AGC CGCAAAGTGTGTAACGGAA RKVCNGIGIGEFKDSLSINA TAGGTATTGGTGAATTTAA TNIKHFKNCTSISGDLHILP AGACTCACTCTCCATAAAT VAFRGDSFTHTPPLDPQEL EGFR truncated GCTACGAATATTAAACACT DILKTVKEITGFLLIQAWPE design 5 (Herd TCAAAAACTGCACCTCCAT NRTDLHAFENLEIIRGRTK design5(Hin 81 CAGTGGCGATCTCCACATC 194 QHGQFSLAVVSLNITSLGL truc teddesign CTGCCGGTGGCATTTAGGG RSLKEISDGDVIISGNKNLC GTGACTCCTTCACACATACT YANTINWKKLFGTSGQKT CCTCCTCTGGATCCACAGG KIISNRGENSCKATGQVCH AACTGGATATTCTGAAAAC ALCSPEGCWGPEPRDCVSC CGTAAAGGAAATCACAGGG RNVSRGRECVDKCNLLEG

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO TTTTTGCTGATTCAGGCTTG EPREFVENSECIQCHPECLP GCCTGAAAACAGGACGGAC QAMNITCTGRGPDNCIQCA CTCCATGCCTTTGAGAACCT HYIDGPHCVKTGGGGSGG AGAAATCATACGCGGCAGG GGSGGGGSFWVLVVVGGV ACCAAGCAACATGGTCAGT LACYSLLVTVAFIIFWVRS TTTCTCTTGCAGTCGTCAGC KRS CTGAACATAACATCCTTGG GATTACGCTCCCTCAAGGA GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCTT GCCGGAATGTCAGCCGAGG CAGGGAATGCGTGGACAAG TGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGA GAACTCTGAGTGCATACAG TGCCACCCAGAGTGCCTGC CTCAGGCCATGAACATCAC CTGCACAGGACGGGGACCA GACAACTGTATCCAGTGTG CCCACTACATTGACGGCCC CCACTGCGTCAAGACCGGC GGAGGCGGAAGCGGAGGC GGAGGCTCCGGCGGAGGCG GAAGCTTTTGGGTGCTGGT GGTGGTTGGTGGAGTCCTG GCTTGCTATAGCTTGCTAGT AACAGTGGCCTTTATTATTT TCTGGGTGAGGAGTAAGAG GAGC CGCAAAGTGTGTAACGGAA RKVCNGIGIGEFKDSLSINA TAGGTATTGGTGAATTTAA TNIKHFKNCTSISGDLHILP EGFR truncated AGACTCACTCTCCATAAAT VAFRGDSFTHTPPLDPQEL GCTACGAATATTAAACACT DILKTVKEITGFLLIQAWPE des design 82 TCAAAAACTGCACCTCCAT 195 NRTDLHAFENLEIIRGRTK truncatdsg CAGTGGCGATCTCCACATC QHGQFSLAVVSLNITSLGL 6)(~HERlt6) CTGCCGGTGGCATTTAGGG RSLKEISDGDVIISGNKNLC GTGACTCCTTCACACATACT YANTINWKKLFGTSGQKT CCTCCTCTGGATCCACAGG KIISNRGENSCKATGQVCH

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO AACTGGATATTCTGAAAAC ALCSPEGCWGPEPRDCVSC CGTAAAGGAAATCACAGGG RNVSRGRECVDKCNLLEG TTTTTGCTGATTCAGGCTTG EPREFVENSECIQCHPECLP GCCTGAAAACAGGACGGAC QAMNITCTGRGPDNCIQCA CTCCATGCCTTTGAGAACCT HYIDGPHCVKTCPAGVMG AGAAATCATACGCGGCAGG ENNTLVWKYADAGHVCH ACCAAGCAACATGGTCAGT LGGGGSGGGGSFWVLVVV TTTCTCTTGCAGTCGTCAGC GGVLACYSLLVTVAFIIFW CTGAACATAACATCCTTGG VRSKRS GATTACGCTCCCTCAAGGA GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCTT GCCGGAATGTCAGCCGAGG CAGGGAATGCGTGGACAAG TGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGA GAACTCTGAGTGCATACAG TGCCACCCAGAGTGCCTGC CTCAGGCCATGAACATCAC CTGCACAGGACGGGGACCA GACAACTGTATCCAGTGTG CCCACTACATTGACGGCCC CCACTGCGTCAAGACCTGC CCGGCAGGAGTCATGGGAG AAAACAACACCCTGGTCTG GAAGTACGCAGACGCCGGC CATGTGTGCCACCTGGGCG GAGGCGGAAGCGGAGGCG GAGGCTCCTTTTGGGTGCTG GTGGTGGTTGGTGGAGTCC TGGCTTGCTATAGCTTGCTA GTAACAGTGGCCTTTATTAT TTTCTGGGTGAGGAGTAAG AGGAGC EGFR truncated CGCAAAGTGTGTAACGGAA RKVCNGIGIGEFKDSLSINA design 7 (Herl 83 TAGGTATTGGTGAATTTAA 196 TNIKHFKNCTSISGDLHILP truncated design AGACTCACTCTCCATAAAT VAFRGDSFTHTPPLDPQEL 7) (HERlt7) GCTACGAATATTAAACACT DILKTVKEITGFLLIQAWPE

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO TCAAAAACTGCACCTCCAT NRTDLHAFENLEIIRGRTK CAGTGGCGATCTCCACATC QHGQFSLAVVSLNITSLGL CTGCCGGTGGCATTTAGGG RSLKEISDGDVIISGNKNLC GTGACTCCTTCACACATACT YANTINWKKLFGTSGQKT CCTCCTCTGGATCCACAGG KIISNRGENSCKATGQVCH AACTGGATATTCTGAAAAC ALCSPEGCWGPEPRDCVSC CGTAAAGGAAATCACAGGG RNVSRGRECVDKCNLLEG TTTTTGCTGATTCAGGCTTG EPREFVENSECIQCHPECLP GCCTGAAAACAGGACGGAC QAMNITCTGRGPDNCIQCA CTCCATGCCTTTGAGAACCT HYIDGPHCVKTCPAGVMG AGAAATCATACGCGGCAGG ENNTLVWKYADAGHVCH ACCAAGCAACATGGTCAGT LCHPNCTYGCTGPGLEGCP TTTCTCTTGCAGTCGTCAGC GGGGGGSFWVLVVVGGV CTGAACATAACATCCTTGG LACYSLLVTVAFIIFWVRS GATTACGCTCCCTCAAGGA KRS* GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCTT GCCGGAATGTCAGCCGAGG CAGGGAATGCGTGGACAAG TGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGA GAACTCTGAGTGCATACAG TGCCACCCAGAGTGCCTGC CTCAGGCCATGAACATCAC CTGCACAGGACGGGGACCA GACAACTGTATCCAGTGTG CCCACTACATTGACGGCCC CCACTGCGTCAAGACCTGC CCGGCAGGAGTCATGGGAG AAAACAACACCCTGGTCTG GAAGTACGCAGACGCCGGC CATGTGTGCCACCTGTGCCA TCCAAACTGCACCTACGGA TGCACTGGGCCAGGTCTTG AAGGCTGTCCAGGTGGCGG TGGCGGCGGATCTTTTTGGG TGCTGGTGGTGGTTGGTGG AGTCCTGGCTTGCTATAGCT

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO TGCTAGTAACAGTGGCCTTT ATTATTTTCTGGGTGAGGAG TAAGAGGAGCTAA CGCAAAGTGTGTAACGGAA TAGGTATTGGTGAATTTAA AGACTCACTCTCCATAAAT GCTACGAATATTAAACACT TCAAAAACTGCACCTCCAT CAGTGGCGATCTCCACATC CTGCCGGTGGCATTTAGGG GTGACTCCTTCACACATACT CCTCCTCTGGATCCACAGG AACTGGATATTCTGAAAAC CGTAAAGGAAATCACAGGG TTTTTGCTGATTCAGGCTTG GCCTGAAAACAGGACGGAC RKVCNGIGIGEFKDSLSINA CTCCATGCCTTTGAGAACCT TNIKHFKNCTSISGDLHILP AGAAATCATACGCGGCAGG VAFRGDSFTHTPPLDPQEL ACCAAGCAACATGGTCAGT DILKTVKEITGFLLIQAWPE TTTCTCTTGCAGTCGTCAGC NRTDLHAFENLEIIRGRTK EGFR truncated CTGAACATAACATCCTTGG QHGQFSLAVVSLNITSLGL design 8 (Herl 84 GATTACGCTCCCTCAAGGA 197 RSLKEISDGDVIISGNKNLC truncated design GATAAGTGATGGAGATGTG YANTINWKKLFGTSGQKT 8) (HERlt8) ATAATTTCAGGAAACAAAA KIISNRGENSCKATGQVCH ATTTGTGCTATGCAAATACA ALCSPEGCWGPEPRDCVSG ATAAACTGGAAAAAACTGT GGGSGGGGSGGGGSGGGG TTGGGACCTCCGGTCAGAA SEITLIIFGVMAGVIGTILLIS AACCAAAATTATAAGCAAC YGIRRGGGS AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCT GGTGGCGGTGGCTCGGGCG GTGGTGGGTCGGGTGGCGG CGGATCTGGTGGCGGTGGC TCGGAGATAACACTCATTA TTTTTGGGGTGATGGCTGGT GTTATTGGAACGATCCTCTT AATTTCTTACGGTATTCGCC GAGGAGGTGGAAGC CGCAAAGTGTGTAACGGAA RKVCNGIGIGEFKDSLSINA EGFR truncated TAGGTATTGGTGAATTTAA TNIKHFKNCTSISGDLHILP design 9 (Herl 85 AGACTCACTCTCCATAAAT 198 VAFRGDSFTHTPPLDPQEL truncated design GCTACGAATATTAAACACT DILKTVKEITGFLLIQAWPE 9) (HERlt9) TCAAAAACTGCACCTCCAT NRTDLHAFENLEIIRGRTK CAGTGGCGATCTCCACATC QHGQFSLAVVSLNITSLGL

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO CTGCCGGTGGCATTTAGGG RSLKEISDGDVIISGNKNLC GTGACTCCTTCACACATACT YANTINWKKLFGTSGQKT CCTCCTCTGGATCCACAGG KIISNRGENSCKATGQVCH AACTGGATATTCTGAAAAC ALCSPEGCWGPEPRDCVSG CGTAAAGGAAATCACAGGG GGGSGGGGSGGGGSGGGG TTTTTGCTGATTCAGGCTTG SITLIIFGVMAGVIGTILLIS GCCTGAAAACAGGACGGAC YGIGGGS CTCCATGCCTTTGAGAACCT AGAAATCATACGCGGCAGG ACCAAGCAACATGGTCAGT TTTCTCTTGCAGTCGTCAGC CTGAACATAACATCCTTGG GATTACGCTCCCTCAAGGA GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCT GGTGGCGGTGGCTCGGGCG GTGGTGGGTCGGGTGGCGG CGGATCTGGTGGCGGTGGC TCGATAACACTCATTATTTT TGGGGTGATGGCTGGTGTT ATTGGAACGATCCTCTTAAT TTCTTACGGTATTGGAGGTG GAAGC CGCAAAGTGTGTAACGGAA RKVCNGIGIGEFKDSLSINA TAGGTATTGGTGAATTTAA TNIKHFKNCTSISGDLHILP AGACTCACTCTCCATAAAT VAFRGDSFTHTPPLDPQEL GCTACGAATATTAAACACT DILKTVKEITGFLLIQAWPE TCAAAAACTGCACCTCCAT NRTDLHAFENLEIIRGRTK EGFR truncated CAGTGGCGATCTCCACATC QHGQFSLAVVSLNITSLGL design 10 (Herl CTGCCGGTGGCATTTAGGG RSLKEISDGDVIISGNKNLC truncated design 86 GTGACTCCTTCACACATACT 199 YANTINWKKLFGTSGQKT 10) (HERlt1O) CCTCCTCTGGATCCACAGG KIISNRGENSCKATGQVCH AACTGGATATTCTGAAAAC ALCSPEGCWGPEPRDCVSG CGTAAAGGAAATCACAGGG GGGSGGGGSGGGGSGGGG TTTTTGCTGATTCAGGCTTG SITLIIFGVMAGVIGTILLAL GCCTGAAAACAGGACGGAC LIWGGGS CTCCATGCCTTTGAGAACCT AGAAATCATACGCGGCAGG

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO ACCAAGCAACATGGTCAGT TTTCTCTTGCAGTCGTCAGC CTGAACATAACATCCTTGG GATTACGCTCCCTCAAGGA GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCT GGTGGCGGTGGCTCGGGCG GTGGTGGGTCGGGTGGCGG CGGATCTGGTGGCGGTGGC TCGATAACACTCATTATTTT TGGGGTGATGGCTGGTGTT ATTGGAACGATCCTCTTAGC CCTGCTCATCTGGGGAGGT GGAAGC CGCAAAGTGTGTAACGGAA TAGGTATTGGTGAATTTAA AGACTCACTCTCCATAAAT GCTACGAATATTAAACACT TCAAAAACTGCACCTCCAT CAGTGGCGATCTCCACATC RKVCNGIGIGEFKDSLSINA CTGCCGGTGGCATTTAGGG TNIKHFKNCTSISGDLHILP GTGACTCCTTCACACATACT VAFRGDSFTHTPPLDPQEL CCTCCTCTGGATCCACAGG DILKTVKEITGFLLIQAWPE AACTGGATATTCTGAAAAC NRTDLHAFENLEIIRGRTK EGFR truncated CGTAAAGGAAATCACAGGG QHGQFSLAVVSLNITSLGL design 11 (Herl 87 TTTTTGCTGATTCAGGCTTG 200 RSLKEISDGDVIISGNKNLC truncated design GCCTGAAAACAGGACGGAC YANTINWKKLFGTSGQKT 11) (HERlt11) CTCCATGCCTTTGAGAACCT KIISNRGENSCKATGQVCH AGAAATCATACGCGGCAGG ALCSPEGCWGPEPRDCVSG ACCAAGCAACATGGTCAGT GGGSGGGGSGGGGSGGGG TTTCTCTTGCAGTCGTCAGC SLCYLLDGILFIYGVILTAL CTGAACATAACATCCTTGG FLGGGS GATTACGCTCCCTCAAGGA GATAAGTGATGGAGATGTG ATAATTTCAGGAAACAAAA ATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGT TTGGGACCTCCGGTCAGAA

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO AACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTG CCATGCCTTGTGCTCCCCCG AGGGCTGCTGGGGCCCGGA GCCCAGGGACTGCGTCTCT GGTGGCGGTGGCTCGGGCG GTGGTGGGTCGGGTGGCGG CGGATCTGGTGGCGGTGGC TCGCTCTGCTACCTGCTGGA TGGAATCCTCTTCATCTATG GTGTCATTCTCACTGCCTTG TTCCTGGGAGGTGGAAGC ATGACAACACCCAGAAATT CAGTAAATGGGACTTTCCC GGCAGAGCCAATGAAAGGC CCTATTGCTATGCAATCTGG TCCAAAACCACTCTTCAGG AGGATGTCTTCACTGGTGG GCCCCACGCAAAGCTTCTTC ATGAGGGAATCTAAGACTT TGGGGGCTGTCCAGATTAT MTTPRNSVNGTFPAEPMK GAATGGGCTCTTCCACATTG GPIAMQSGPKPLFRRMSSL CCCTGGGGGGTCTTCTGATG VGPTQSFFMRESKTLGAVQ ATCCCAGCAGGGATCTATG IMNGLFHIALGGLLMIPAGI CACCCATCTGTGTGACTGTG YAPICVTVWYPLWGGIMYI TGGTACCCTCTCTGGGGAG ISGSLLAATEKNSRKCLVK GCATTATGTATATTATTTCC GKMIMNSLSLFAAISGMILS GGATCACTCCTCGGCAGCAA IMDILNIKISHFLKMESLNFI FL CD20 88 CGGAGAAAAACTCCAGGAA 201 RAHTPYINIYNCEPANPSEK GTGTTTGGTCAAAGGAAAA NSPSTQYCYSIQSLFLGILS ATGATAATGAATTCATTGA VMLIFAFFQELVIAGIVENE GCCTCTTTGCTGCCATTTCT WKRTCSRPKSNIVLLSAEE GGAATGATTCTTTCAATCAT KKEQTIEIKEEVVGLTETSS GGACATACTTAATATTAAA QPKNEEDIEIIPIQEEEEEET ATTTCCCATTTTTTAAAAAT ETNFPEPPQDQESSPIENDS GGAGAGTCTGAATTTTATTA GAGCTCACACACCATATAT TAACATATACAACTGTGAA CCAGCTAATCCCTCTGAGA AAAACTCCCCATCTACCCA ATACTGTTACAGCATACAA TCTCTGTTCTTGGGCATTTT GTCAGTGATGCTGATCTTTG CCTTCTTCCAGGAACTTGTA ATAGCTGGCATCGTTGAGA ATGAATGGAAAAGAACGTG

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO CTCCAGACCCAAATCTAAC ATAGTTCTCCTGTCAGCAGA AGAAAAAAAAGAACAGACT ATTGAAATAAAAGAAGAAG TGGTTGGGCTAACTGAAAC ATCTTCCCAACCAAAGAAT GAAGAAGACATTGAAATTA TTCCAATCCAAGAAGAGGA AGAAGAAGAAACAGAGAC GAACTTTCCAGAACCTCCCC AAGATCAGGAATCCTCACC AATAGAAAATGACAGCTCT CCT ATGACCACACCACGGAACT CTGTGAATGGCACCTTCCCA GCAGAGCCAATGAAGGGAC CAATCGCAATGCAGAGCGG ACCCAAGCCTCTGTTTCGGA GAATGAGCTCCCTGGTGGG CCCAACCCAGTCCTTCTTTA TGAGAGAGTCTAAGACACT GGGCGCCGTGCAGATCATG AACGGACTGTTCCACATCG CCCTGGGAGGACTGCTGAT MTTPRNSVNGTFPAEPMK GATCCCAGCCGGCATCTAC GPIAMQSGPKPLFRRMSSL GCCCCTATCTGCGTGACCGT VGPTQSFFMRESKTLGAVQ GTGGTACCCTCTGTGGGGC IMNGLFHIALGGLLMIPAGI GGCATCATGTATATCATCTC YAPICVTVWYPLWGGIMYI Truncated CD20 CGGCTCTCTGCTGGCCGCCA ISGSLLAATEKNSRKCLVK design 1 89 CAGAGAAGAACAGCAGGA 202 GKMIMNSLSLFAAISGMILS (CD20tl) AGTGTCTGGTGAAGGGCAA IMDILNIKISHFLKMESLNFI

[CD20(M1-E263] GATGATCATGAATAGCCTG RAHTPYINIYNCEPANPSEK TCCCTGTTTGCCGCCATCTC NSPSTQYCYSIQSLFLGILS TGGCATGATCCTGAGCATC VMLIFAFFQELVIAGIVENE ATGGACATCCTGAACATCA WKRTCSRPKSNIVLLSAEE AGATCAGCCACTTCCTGAA KKEQTIEIKEEVVGLTETSS GATGGAGAGCCTGAACTTC QPKNEEDIE ATCAGAGCCCACACCCCTT ACATCAACATCTATAATTGC GAGCCTGCCAACCCATCCG AGAAGAATTCTCCAAGCAC ACAGTACTGTTATTCCATCC AGTCTCTGTTCCTGGGCATC CTGTCTGTGATGCTGATCTT TGCCTTCTTTCAGGAGCTGG TCATCGCCGGCATCGTGGA GAACGAGTGGAAGAGGACC

SEQ SEQ Polynucleotide Sequence (5' to Amino Acid Sequence (5' to Cell Tag Name ID ID 3' where applicable) 3' where applicable) NO NO TGCAGCCGCCCCAAGTCCA ATATCGTGCTGCTGTCCGCC GAGGAGAAGAAGGAGCAG ACAATCGAGATCAAGGAGG AGGTGGTGGGCCTGACCGA GACATCTAGCCAGCCTAAG AATGAGGAGGATATCGAG

[0337] In further embodiments, at least one of said gene expression cassettes comprises a polynucleotide encoding a promoter that is activated by the transactivation domain. In further embodiments, said system is contained in one or more vectors. In one instance, the system is contained in one vector. In one instance, the first gene expression cassette, the second gene expression cassette, and the recombinant attachment sites are contained in one vector. In one instance, the first gene expression cassette, the second gene expression cassette, the third gene expression cassette and the recombinant attachment sites are contained in one vector. In another instance, the serine recombinase is SF370. In other instances, the serine recombinase is in a separate vector.

[0338] The recombinases can be introduced into a target cell before, concurrently with, or after the introduction of a targeting vector. The recombinase can be directly introduced into a cell as a protein, for example, using liposomes, coated particles, or microinjection. Alternately, a polynucleotide, either DNA or messenger RNA, encoding the recombinase can be introduced into the cell using a suitable expression vector. The targeting vector components described above are useful in the construction of expression cassettes containing sequences encoding a recombinase of interest. However, expression of the recombinase can be regulated in other ways, for example, by placing the expression of the recombinase under the control of a regulatable promoter (i.e., a promoter whose expression can be selectively induced or repressed).

[0339] Recombinases for use in the practice of the present disclosure can be produced recombinantly or purified as previously described. Polypeptides having the desired recombinase activity can be purified to a desired degree of purity by methods known in the art of protein ammonium sulfate precipitation, purification, including, but not limited to, size fractionation, affinity chromatography, HPLC, ion exchange chromatography, heparin agarose affinity chromatography (e.g., Thorpe & Smith, Proc. Nat. Acad. Sci. 95:5505-5510, 1998.)

[0340] In one embodiment, the recombinases can be introduced into the eukaryotic cells that contain the recombination attachment sites at which recombination is desired by any suitable method. Methods of introducing functional proteins, e.g., by microinjection or other methods, into cells are well known in the art. Introduction of purified recombinase protein ensures a transient presence of the protein and its function, which is often a preferred embodiment. Alternatively, a gene encoding the recombinase can be included in an expression vector used to transform the cell, in which the recombinase-encoding polynucleotide is operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell. The recombinase polypeptide can also be introduced into the eukaryotic cell by messenger RNA that encodes the recombinase polypeptide. It is generally preferred that the recombinase be present for only such time as is necessary for insertion of the nucleic acid fragments into the genome being modified. Thus, the lack of permanence associated with most expression vectors is not expected to be detrimental. One can introduce the recombinase gene into the cell before, after, or simultaneously with, the introduction of the exogenous polynucleotide of interest. In one embodiment, the recombinase gene is present within the vector that carries the polynucleotide that is to be inserted; the recombinase gene can even be included within the polynucleotide. In other embodiments, the recombinase gene is introduced into a transgenic eukaryotic organism. Transgenic cells or animals can be made that express a recombinase constitutively or under cell specific, tissue-specific, developmental-specific, organelle-specific, or small molecule-inducible or repressible promoters. The recombinases can be also expressed as a fusion protein with other peptides, proteins, nuclear localizing signal peptides, signal peptides, or organelle-specific signal peptides (e.g., mitochondrial or chloroplast transit peptides to facilitate recombination in mitochondria or chloroplast).

[0341] For example, a recombinase may be from the Integrase or Resolvase families. The Integrase family of recombinases has over one hundred members and includes, for example, FLP, Cre, and lambda integrase. The Integrase family, also referred to as the tyrosine family or the lambda integrase family, uses the catalytic tyrosine's hydroxyl group for a nucleophilic attack on the phosphodiester bond of the DNA. Typically, members of the tyrosine family initially nick the DNA, which later forms a double strand break. Examples of tyrosine family integrases include Cre, FLP, SSV1, and lambda (k) integrase. In the resolvase family, also known as the serine recombinase family, a conserved serine residue forms a covalent link to the DNA target site (Grindley, et al., (2006) Ann Rev Biochem 16:16).

[0342] In one embodiment, the recombinase is an isolated polynucleotide sequence comprising a nucleic acid sequence that encodes a recombinase selecting from the group consisting of a SPjc2 recombinase, a SF370.1 recombinase, a Bxbl recombinase, an Al18 recombinase and a <}Rvl recombinase. Examples of serine recombinases are described in detail in U.S. Patent No. 9,034,652, hereby incorporated by reference in its entirety.

[0343] In one embodiment, a method for site-specific recombination comprises providing a first recombination site and a second recombination site; contacting the first and second recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites, wherein the recombinase polypeptide can mediate recombination between the first and second recombination sites, the first recombination site is attP or attl, the second recombination site is attf or attP, and the recombinase is selected from the group consisting of a Listeriamonocytogenes phage recombinase, a Streptococcuspyogenes phage recombinase, a Bacillus subtilis phage recombinase, aMycobacterium tuberculosisphage recombinase and a Mycobacterium smegmatis phage recombinase, provided that when the first recombination attachment site is att, the second recombination attachment site is attP, and when the first recombination attachment site is attP, the second recombination attachment site is attl Further embodiments provide for the introduction of a site-specific recombinase into a cell whose genome is to be modified. One embodiment relates to a method for obtaining site-specific recombination in an eukaryotic cell comprises providing a eukaryotic cell that comprises a first recombination attachment site and a second recombination attachment site; contacting the first and second recombination attachment sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination attachment sites, wherein the recombinase polypeptide can mediate recombination between the first and second recombination attachment sites, the first recombination attachment site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination attachment site is attf or attP, and the recombinase is selected from the group consisting of a Listeriamonocytogenes phage recombinase, a Streptococcuspyogenes phage recombinase, a Bacillus subtilis phage recombinase, a Mycobacterium tuberculosis phage recombinase and a Mycobacterium smegmatis phage recombinase, provided that when the first recombination attachment site is attl, the second recombination attachment site is attP, and when the first recombination attachment site is attP, the second recombination attachment site is attl. In an embodiment the recombinase is selected from the group consisting of an Al18 recombinase, a SF370.1 recombinase, a SP c2 recombinase, a c Rvl recombinase, and a Bxbl recombinase. In one embodiment the recombination results in integration.

Immune Effector Cell Sources

[0344] In certain aspects, the embodiments described herein include methods of making and/or expanding the antigen-specific redirected immune effector cells (e.g., T-cells, NK-cell or NK T cells) that comprises transfecting the cells with an expression vector containing a DNA (or RNA) construct encoding the CAR, then, optionally, stimulating the cells with feeder cells, recombinant antigen, or an antibody to the receptor to cause the cells to proliferate. In certain aspects, the cell (or cell population) engineered to express a CAR or TCR is a stem cell, iPS cell, immune effector cell or a precursor of these cells.

[0345] Sources of immune effector cells can include both allogeneic and autologous sources. In some cases immune effector cells may be differentiated from stem cells or induced pluripotent stem cells (iPSCs). Thus, cells for engineering according to the embodiments can be isolated from umbilical cord blood, peripheral blood, human embryonic stem cells, or iPSCs. For example, allogeneic T cells can be modified to include a chimeric antigen receptor (and optionally, to lack functional TCR). In some aspects, the immune effector cells are primary human T cells such as T cells derived from human peripheral blood mononuclear cells (PBMC). PBMCs can be collected from the peripheral blood or after stimulation with G-CSF (Granulocyte colony stimulating factor) from the bone marrow, or umbilical cord blood. Following transfection or transduction (e.g., with a CAR expression construct), the cells may be immediately infused or may be cryo-preserved. In certain aspects, following transfection, the cells may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days or more following gene transfer into cells. In a further aspect, following transfection, the transfectants are cloned and a clone demonstrating presence of a single integrated or episomally maintained expression cassette or plasmid, and expression of the chimeric antigen receptor is expanded ex vivo. The clone selected for expansion demonstrates the capacity to specifically recognize and lyse antigen-expressing target cells. The recombinant T cells may be expanded by stimulation with IL-2, or other cytokines that bind the common gamma-chain (e.g., L-7, IL-12, IL-15, IL-21, and others). The recombinant T cells may be expanded by stimulation with artificial antigen presenting cells. The recombinant T cells may be expanded on artificial antigen presenting cell or with an antibody, such as OKT3, which cross links CD3 on the T cell surface. Subsets of the recombinant T cells may be further selected with the use of magnetic bead based isolation methods and/or fluorescence activated cell sorting technology and further cultured with the AaPCs. In a further aspect, the genetically modified cells may be cryopreserved.

[0346] T cells can also be obtained from a number of sources, including peripheral blood, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumor (tumor-infiltrating lymphocytes). In certain embodiments of the present disclosure, any number of T cell lines available in the art, may be used. In certain embodiments of the present disclosure, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll@ separation. In embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the present disclosure, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0347] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL@ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3x28)-conjugated beads, such as DYNABEADS@ M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune-compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this disclosure. In certain embodiments, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

[0348] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1Ib, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar method of selection.

[0349] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28 negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[0350] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5x106/ml. In other embodiments, the concentration used can be from about 1x105/ml to 1x106/ml, and any integer value in between.

[0351] In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10o C or at room temperature.

[0352] T cells for stimulation can also be frozen after a washing step. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7 .5

% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80o C at a rate of 1 oper minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20o C or in liquid nitrogen.

[0353] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.

[0354] Also contemplated in the context of the present disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66:807 815, (1991); Henderson et al., Immun 73:316-321, (1991); Bierer et al., Curr. Opin. Immun 5:763-773, (1993)). In a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.

[0355] In a further embodiment of the present disclosure, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Activation and Expansion of T Cells

[0356] In certain embodiments are T cells comprising polynucleotides encoding gene-switch polypeptides for expressing an interleukin, CARs and/or TCRs described herein Whether prior to or after genetic modification of the T cells to express a desirable CAR, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;

7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0357] "Adoptive T cell transfer" refers to the isolation and ex vivo or in vivo expansion of tumor specific T cells to achieve greater number of T cells than what could be obtained by vaccination alone or the patient's natural tumor response. The tumor specific T cells are then infused into patients with cancer in an attempt to give their immune system the ability to overwhelm remaining tumor via T cells which can attack and kill cancer. There are many forms of adoptive T cell therapy being used for cancer treatment; culturing tumor infiltrating lymphocytes or TIL, isolating and expanding one particular T cell or clone, and even using T cells that have been engineered to potently recognize and attack tumors. In some aspects, adoptive T-cell therapy can include engineered antigen-specific T cells that are modified and immediately infused into a patient, thus allowing in vivo expansion of antigen-specific T cells to occur within the patient.

[0358] In some cases, T cells described herein are activated and/or expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, (1998); Haanen et al., J. Exp. Med. 190(9):13191328, (1999); Garland et al., J. Immunol Meth. 227(1 2):53-63, (1999)).

[0359] In certain embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (AaPC) that are contemplated for use in activating and expanding T cells in the present disclosure.

[0360] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., "cis," or to separate beads, i.e., "trans." By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1:1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain aspects of the present disclosure, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1:1. In one particular embodiment an increase of from about I to about 3 fold is observed as compared to the expansion observed using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between. In one aspect of the present disclosure, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the present disclosure, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1. In one particular embodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In embodiments, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

[0361] Ratios of particles to cells from 1:500 to 500:1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1:100 to 100:1 and any integer values in-between and in further embodiments the ratio comprises 1:9 to 9:1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28 coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1 with one ratio being at least 1:1 particles per T cell. In one embodiment, a ratio of particles to cells of 1:1 or less is used. In one particular embodiment, the particle:cell ratio is 1:5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1:1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present disclosure. In particular, ratios will vary depending on particle size and on cell size and type.

[0362] In further embodiments of the present disclosure, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0363] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells. In one embodiment the cells (for example, 104 to 109 T cells) and beads (for example, DYNABEADS@ M-450 CD3/CD28 T paramagnetic beads at a ratio of 1:1, or MACS® MicroBeads from Miltenyi Biotec) are combined in a buffer, for example, PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present disclosure. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[0364] In one embodiment of the present disclosure, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the present disclosure the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-.gamma., L-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFbeta, and TNF alpha or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37o C.) and atmosphere (e.g., air plus 5% C02).

[0365] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and

CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

[0366] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

[0367] In some cases, immune effector cells of the embodiments (e.g., T-cells) are co-cultured with activating and propagating cells (AaPCs), to aid in cell expansion. AaPCs can also be referred to as artificial Antigen Presenting cells (AaPCs). For example, antigen presenting cells (APCs) are useful in preparing therapeutic compositions and cell therapy products of the embodiments. In one aspect, the AaPCs may be transgenic K562 cells. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application Publication Nos. 2009/0017000 and 2009/0004142; and International Publication No. W02007/103009, each of which is incorporated by reference. In yet a further aspect of the embodiments, culturing the transgenic CAR cells comprises culturing the transgenic CAR cells in the presence of dendritic cells or activating and propagating cells (AaPCs) that stimulate expansion of the CAR-expressing immune effector cells. In still further aspects, the AaPCs comprise a CAR-binding antibody or fragment thereof expressed on the surface of the AaPCs. In other embodiments, the AaPCS comprise a TCR-binding polypeptide or TCR binding antibody or fragment thereof expressed on the surface of the AaPCs. The AaPCs may comprise additional molecules that activate or co stimulate T-cells in some cases. The additional molecules may, in some cases, comprise membrane-bound Cycytokines. In yet still further aspects, the AaPCs are inactivated or irradiated, or have been tested for and confirmed to be free of infectious material. In still further aspects, culturing the transgenic CAR cells in the presence of AaPCs comprises culturing the transgenic CAR cells in a medium comprising soluble cytokines, such as IL-15, IL-21 and/or IL 2. The cells maybe cultured at a ratio of about 10:1 to about 1:10; about 3:1 to about 1:5; about 1:1 to about 1:3 (immune effector cells to AaPCs); or any range derivable therein. For example, the co-culture of T cells and AaPCs can be at a ratio of about 1:1, about 1:2 or about 1:3.

[0368] In one aspect, the AaPCs may express CD137L. In other aspects, the AaPCs may further express CD19, CD64, CD86, or mIL15. In certain aspects, the AaPCs may express at least one anti-CD3 antibody clone, such as, for example, OKT3 and/or UCHT1. In one aspect, the AaPCs may be treated (e.g. irradiated or mytomycin C) to eliminate their growth potential. In one aspect, the AaPCs may have been tested for and confirmed to be free of infectious material. Methods for producing such AaPCs are known in the art. In one aspect, culturing the CAR modified T cell population with AaPCs may comprise culturing the cells at a ratio of about 10:1 to about 1:10; about 3:1 to about 1:5; about 1:1 to about 1:3 (T cells to AaPCs); or any range derivable therein. For example, the co-culture of T cells and AaPCs can be at a ratio of about 1:1, about 1:2 or about 1:3. In one aspect, the culturing step may further comprise culturing with an aminobisphosphonate (e.g., zoledronic acid).

[0369] In a further aspect, the population of CAR-T cells is cultured and/or stimulated for no more than 7, 14, 21, 28, 35 42 days, 49, 56, 63 or 70 days. In some embodiments, the population of CAR-T cells is cultured and/or stimulated for at least 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or more days. In some embodiments, the population of CAR-T cells is cultured and/or stimulated for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days. In some embodiments, the population of CAR-T cells is cultured and/or stimulated for at least 7, 14, 21, 28, 35, 42, 49, 56, 63 or more days. In other embodiments, a stimulation includes the co-culture of the CAR-T cells with AaPCs to promote the growth of CAR positive T cells. In another aspect, the population of transgenic CAR cells is stimulated for not more than: 1X stimulation, 2X stimulation, 3X stimulation, 4X stimulation, 5X stimulation, 5X stimulation, 6X stimulation, 7X stimulation, 8X stimulation, 9X stimulation or lOX stimulation. In some instances, the transgenic cells are not cultured ex vivo in the presence of AaPCs. In some specific instances, the method of the embodiment further comprises enriching the cell population for CAR-expressing immune effector cells (e.g., T-cells) after the transfection and/or culturing step. The enriching may comprise fluorescence-activated cell sorting (FACS) and sorting for CAR-expressing cells. In a further aspect, the sorting for CAR-expressing cells comprises use of a CAR-binding antibody. The enriching may also comprise depletion of CD56+ cells. In yet still a further aspect of the embodiment, the method further comprises cryopreserving a sample of the population of transgenic CAR cells.

[0370] In some cases, AaPCs are incubated with a peptide of an optimal length that allows for direct binding of the peptide to the MIIC molecule without additional processing. Alternatively, the cells can express an antigen of interest (i.e., in the case of MC-independent antigen recognition). Furthermore, in some cases, APCs can express an antibody that binds to either a specific CAR polypeptide or to CAR polypeptides in general (e.g., a universal activating and propagating cell (uAPC). Such methods are disclosed in WO/2014/190273, which is incorporated herein by reference. In addition to peptide-MHC molecules or antigens of interest, the AaPC systems may also comprise at least one exogenous assisting molecule. Any suitable number and combination of assisting molecules may be employed. The assisting molecule may be selected from assisting molecules such as co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD70 and B7.1 (B7.1 was previously known as B7 and also known as CD80), which among other things, bind to CD28 and/or CTLA-4 molecules on the surface of T cells, thereby affecting, for example, T-cell expansion, Th1 differentiation, short-term T-cell survival, and cytokine secretion such as interleukin (IL)-2. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), that promote, for example, cell-to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selection, cloning, preparation, and expression of exemplary assisting molecules, including co-stimulatory molecules and adhesion molecules, are exemplified in, e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001, incorporated herein by reference.

[0371] Cells selected to become AaPCs, preferably have deficiencies in intracellular antigen processing, intracellular peptide trafficking, and/or intracellular MHC Class I or Class II molecule-peptide loading, or are poikilothermic (i.e., less sensitive to temperature challenge than mammalian cell lines), or possess both deficiencies and poikilothermic properties. Preferably, cells selected to become AaPCs also lack the ability to express at least one endogenous counterpart (e.g., endogenous MC Class I or Class II molecule and/or endogenous assisting molecules as described above) to the exogenous MHC Class I or Class II molecule and assisting molecule components that are introduced into the cells. Furthermore, AaPCs preferably retain the deficiencies and poikilothermic properties that were possessed by the cells prior to their modification to generate the AaPCs. Exemplary AaPCs either constitute or are derived from a transporter associated with antigen processing (TAP)-deficient cell line, such as an insect cell line. An exemplary poikilothermic insect cells line is a Drosophila cell line, such as a Schneider 2 cell line (see, e.g., Schneider 1972 Illustrative methods for the preparation, growth, and culture of Schneider 2 cells, are provided in U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.

[0372] In one embodiment, AaPCs are also subjected to a freeze-thaw cycle. In an exemplary freeze-thaw cycle, the AaPCs may be frozen by contacting a suitable receptacle containing the AaPCs with an appropriate amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or similar low-temperature material, such that freezing occurs rapidly. The frozen APCs are then thawed, either by removal of the AaPCs from the low-temperature material and exposure to ambient room temperature conditions, or by a facilitated thawing process in which a lukewarm water bath or warm hand is employed to facilitate a shorter thawing time. Additionally, AaPCs may be frozen and stored for an extended period of time prior to thawing. Frozen AaPCs may also be thawed and then lyophilized before further use. Preferably, preservatives that might detrimentally impact the freeze-thaw procedures, such as dimethyl sulfoxide (DMSO), polyethylene glycols (PEGs), and other preservatives, are absent from media containing AaPCs that undergo the freeze-thaw cycle, or are essentially removed, such as by transfer of AaPCs to media that is essentially devoid of such preservatives.

[0373] In further embodiments, xenogenic nucleic acid and nucleic acid endogenous to the AaPCs, may be inactivated by crosslinking, so that essentially no cell growth, replication or expression of nucleic acid occurs after the inactivation. In one embodiment, AaPCs are inactivated at a point subsequent to the expression of exogenous MHC and assisting molecules, presentation of such molecules on the surface of the AaPCs, and loading of presented MC molecules with selected peptide or peptides. Accordingly, such inactivated and selected peptide loaded AaPCs, while rendered essentially incapable of proliferating or replicating, retain selected peptide presentation function. Preferably, the crosslinking also yields AaPCs that are essentially free of contaminating microorganisms, such as bacteria and viruses, without substantially decreasing the antigen-presenting cell function of the AaPCs. Thus crosslinking maintains the important AaPC functions of while helping to alleviate concerns about safety of a cell therapy product developed using the AaPCs. For methods related to crosslinking and AaPCs, see for example, U.S. Patent Application Publication No. 20090017000, which is incorporated herein by reference.

[0374] In certain embodiments there are further provided an engineered antigen presenting cell (APC). Such cells may be used, for example, as described above, to propagate immune effector cells ex vivo. In further aspects, engineered APCs may, themselves be administered to a patient and thereby stimulate expansion of immune effector cells in vivo. Engineered APCs of the embodiments may, themselves, be used as a therapeutic agent. In other embodiments, the engineered APCs can used as a therapeutic agent that can stimulate activation of endogenous immune effector cells specific for a target antigen and/or to increase the activity or persistence of adoptively transferred immune effector cells specific to a target antigen.

[0375] As used herein the term "engineered APC" refers to cell(s) that comprises at least a first transgene, wherein the first transgene encodes a HLA. Such engineered APCs may further comprise a second transgene for expression of an antigen, such that the antigen is presented at the surface on the APC in complex with the HLA. In some aspects, the engineered APC can be a cell type that presented antigens (e.g., a dendritic cell). In further aspects, engineered APC can be produced from a cell type that does not normally present antigens, such a T-cell or T-cell progenitor (referred to as "T-APC"). Thus, in some aspects, an engineered APC of the embodiments comprises a first transgene encoding a target antigen and a second transgene encoding a human leukocyte antigen (HLA), such that the HLA is expressed on the surface of the engineered APC in complex with an epitope of the target antigen. In certain specific aspects, the HLA expressed in the engineered APC is HLA-A2.

[0376] In some aspects, an engineered APC of the embodiments may further comprise at least a third transgene encoding co-stimulatory molecule. The co-stimulatory molecule may be a co stimulatory cytokine that may be a membrane-bound Cy cytokine. In certain aspects, the co stimulatory cytokine is IL-15, such as membrane-bound IL-15. In some further aspects, an engineered APC may comprise an edited (or deleted) gene. For example, an inhibitory gene, such as PD-1, LIM-3, CTLA-4 or a TCR, can be edited to reduce or eliminate expression of the gene. An engineered APC of the embodiments may further comprise a transgene encoding any target antigen of interest. For example, the target antigen can be an infectious disease antigen or a tumor-associated antigen (TAA).

Methods of Regulating Expression

[0377] In one embodiment, a method of regulating the expression of a heterologous gene in an engineered cell is provided. Polynucleotides encoding for gene switch polypeptides for ligand inducible control of a heterologous gene expression, an antigen binding polypeptide and a heterologous gene is provided. In some instances, the polynucleotides are in one or more gene expression cassettes as depicted in any one of Figures 1 through 16. In another instance, the polynucleotides are incorporated into an engineered cell via viral or non-viral vectors. Viral vectors can include lentiviral vectors, retroviral vectors or adenoviral vectors. Non-viral vectors can include Sleeping Beauty transposons. In other instances, the polynucleotides are incorporated into an engineered cell via recombinases or gene editing techniques. Examples of recombinases are serine recombinases as described herein. Examples of gene editing techniques can include CRISPR or Argnonaute systems. Herein a "CRISPR gene editing system" of "CRISPR system" refers to any RNA-guided Cas protein-mediated process for targeting a change in DNA sequence to a specific region of a genome. Herein "Argonaute gene editing system" refers to any single-stranded DNA guided Argonaute endonuclease-mediated process for targeting a change in DNA sequence to a specific region of a genome.

[0378] In some instances, the antigen binding polypeptide can be an antigen binding polypeptide or a ligand binding polypeptide. For example, an antigen binding polypeptide can be an antibody or a fragment thereof, F(ab')2, Fab', Fab, Fv, scFv, variable fragments of heavy chain antibodies (VHHs), a CAR, or an engineered TCR. As a further example, a ligand binding polypeptide can be a receptor. In certain aspects, the heterologous gene is a cytokine. The cytokine can be IL-2, IL-15, IL-12, IL-21, or a fusion of IL-15 and IL-15R (mbIL-15). In certain instances, the cytokine is IL-12. For example, the IL-12 is a single chain IL-12 (scIL-12), protease sensitive IL-12, destabilized IL-12, membrane bound IL-12, intercalated IL-12. In some cases, the heterologous gene is under the control of an inducible promoter. For example, the inducible promoter is a gene switch ligand-inducible promoter for gene transcription.

[0379] In certain instances, the cell is an engineered cell. Herein an engineered cell is a cell which has been modified from its natural or endogenous state. An example of an engineered cell is a cell described herein which has been modified (e.g., by transfection of a polynucleotide into the cell). In some instances, the engineered cell can be an engineered immune effector cell.

[0380] Provided herein is a method to regulate the expression of a heterologous gene wherein the heterologous gene is under the control of a ligand inducible promoter. In some instances, the method results in a low or no basal level of heterologous gene expression in the absence of the ligand. In one instance, the engineered cells are activated prior to inducing the expression of the heterologous gene with a ligand. In another instance, the engineered cells are activated by exposing the cells to an antigen. The antigen can be an antigen that is recognized by the antigen binding polypeptide expressed by the engineered cell. The antigen can be a tumor antigen or an infectious disease antigen.

[0381] The engineered cells can be exposed to the antigen for at least:0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days. In some instances, the engineered cells can be exposed to the antigen for at least:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours.

[0382] In another instance, a population of immune effector cells that have been transduced with the vectors encompassing gene switch polypeptides and inducible gene of interest (described herein in FIG. 2A-2D, FIG. 3,FIG. 19A-19B, FIG. 20A, FIG. 22, FIG. 24A-24D, and FIG. 25A-25B) is cultured and/or stimulated with an engineered AaPC of the embodiments for at least 1X stimulation, 2X stimulation, 3X stimulation or 4X stimulation. In some instances, the engineered AaPC comprises an antigen or a ligand that is recognized by the binding polypeptide expressed on the engineered cells. Following the last stimulation, the cells are allowed to rest before infusion into a patient. The patient is then given the appropriate dose of a ligand used for inducible gene switch regulation to activate and express the gene of interest. In one aspect, the ligand is veledimex. In another aspect, veledimex is provided at 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg. In a further aspect, lower doses of veledimex are provided, for example, 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg or 20 mg.

Point-of-Care

[0383] In one embodiment of the present disclosure, the immune effector cells described herein are modified at a point-of-care site. In some cases, the point-of-care site is at a hospital or at a facility (e.g., a medical facility) near a subject in need of treatment. The subject undergoes apheresis and peripheral blood mononuclear cells (PBMCs) obtained can be enriched for example, by elutriation. In one instance, the elutriation process is performed using a buffer solution containing human serum albumin. Immune effector cells, such as T cells can be isolated by selection methods described herein. In one instance, the selection method for T cells includes beads specific for CD3 and CD8 on T cells. In one case, the beads can be paramagnetic beads. The harvested immune effector cells can be cryopreserved in any appropriate cryopreservation solution prior to modification. The immune effector cells can be thawed up to 24 hours, 36 hours, 48 hours. 72 hours or 96 hours ahead of infusion. The thawed cells can be placed in cell culture buffer, for example in cell culture buffer (e.g. RPMI) supplemented with fetal bovine serum (FBS) or placed in a buffer that includesTL-2 and IL-21, prior to modification. Inanother aspect, the harvested immune effector cells can be modified immediately without the need for cryopreservation.

[0384] In some cases, the immune effector cells are modified by engineering/introducing a chimeric receptor, one or more cell tag(s), and/or cytokine into the immune effector cells and then rapidly infused into a subject. In some cases, the sources of immune effector cells can include both allogeneic and autologous sources. In one case, the immune effector cells can be T cells or NK cells. In one case, the chimeric receptor can be a CAR or a TCR. In another case, the cytokine can be mbIL-15. In one case, the mbIL-15 is of SEQ ID NO:15, or variant or fragment thereof. In yet another case, expression of mbIL-15 is modulated by ligand inducible gene-switch expression systems described herein. For example, a ligand such as veledimex can be delivered to the subject to modulate the expression of mbIL-15. In another aspect, veledimex is provided at 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg. In a further aspect, lower doses of veledimex are provided, for example, 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg or 20 mg. In one embodiment, veledimex is administered to the subject 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days prior to infusion of the modified immune effector cells. In a further embodiment, veledimex is administered about once every 12 hours, about once every 24 hours, about once every 36 hours or about once every48 hours, for an effective period of time to a subject post infusion of the modified immune effector cells. In one embodiment, an effective period of time for veledimex administration is about: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days. In other embodiments, veledimex can be re-administered after a rest period, after a drug holiday or when the subject experiences a relapse.

[0385] In certain cases, where an adverse effect on a subject is observed or when treatment is not needed, the cell tag can be activated, for example via cetuximab, for conditional in vivo ablation of modified immune effector cells comprising cell tags such as truncated epidermal growth factor receptor tags as described herein.

[0386] In some embodiments, such immune effectors cells are modified by the constructs as described in FIG. 2A-2D, FIG. 3,FIG. 19A-19B, FIG. 20A, FIG. 22, FIG. 24A-24D, and FIG. 25A-25B through electroporation. In one instance, electroporation is performed with electroporators such as Lonza's NucleofectorTM electroporators. In other embodiments, the vector comprising the above-mentioned constructs is a non-viral or viral vector. In one case, the non-viral vector includes a Sleeping Beauty transposon-transposase system. In one instance, the immune effector cells are electroporated using a specific sequence. For example, the immune effector cells can be electroporated with one transposon followed by the DNA encoding the transposase followed by a second transposon. In another instance, the immune effector cells can be electroporated with all transposons and transposase at the same time. In another instance, the immune effector cells can be electroporated with a transposase followed by both transposons or one transposon at a time. While undergoing sequential electroporation, the immune effector cells may be rested for a period of time prior to the next electroporation step.

[0387] In some cases, the modified immune effector cells do not undergo a propagation and activation step. In some cases, the modified immune effector cells do not undergo an incubation or culturing step (e.g. ex vivo propagation). In certain cases, the modified immune effector cells are placed in a buffer that includesTL-2 and IL21 prior to infusion. In other instances, the modified immune effector cells are placed or rested in cell culture buffer, for example in cell culture buffer (e.g. RPMI) supplemented with fetal bovine serum (FBS) prior to infusion. Prior to infusion, the modified immune effector cells can be harvested, washed and formulated in saline buffer in preparation for infusion into the subject.

[0388] In one instance, the subject has been lymphodepleted prior to infusion. In other instances, lymphodepletion is not required and the modified immune effector cells are rapidly infused into the subject. Exemplary lymphodepletion regimens are listed in Tables 4 and 5 below:

Table 4. Regimen 1 D-6 Admit / IV Hydration D-5 Fludarabine 25 mg/m2, Cyclophosphamide 250 mg/m2 D-4 Fludarabine 25 mg/m2, Cyclophosphamide 250 mg/m2 D-3 Fludarabine 25 mg/m2 IV, Cyclophosphamide 250 mg/m2 D-2 REST D-1 REST DO T-cell infusion

Table 5. Regimen 2 D-6 Admit / IV Hydration D-5 Fludarabine 30 mg/m2, Cyclophosphamide 500 mg/m2 D-4 Fludarabine 30 mg/m2, Cyclophosphamide 500 mg/m2 D-3 Fludarabine 30 mg/m2 IV, Cyclophosphamide 500 mg/m2 D-2 REST D-1 REST DO T-cell infusion

[0389] In a further instance, the subject undergoes minimal lymphodepletion. Minimal lymphodepletion herein refers to a reduced lymphodepletion protocol such that the subject can be infused within 1 day, 2 days or 3 days following the lymphodepletion regimen. In one instance, a reduced lymphodepletion protocol can include lower doses of fludarabine and/or cyclophosphamide. In another instance, a reduced lymphodepetion protocol can include a shortened period of lymphodepletion, for example 1 day or 2 days.

[0390] In one embodiment, the immune effector cells are modified by engineering/introducing a chimeric receptor and a cytokine into said immune effector cells and then rapidly infused into a subject. In other cases, the immune effector cells are modified by engineering/introducing a chimeric receptor and a cytokine into said cells and then infused within at least: 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 hours into a subject. In other cases, immune effector cells are modified by engineering/introducing a chimeric receptor and a cytokine into the immune effector cells and then infused in 0 days, <1 day, <2 days, <3 days, <4 days, <5 days, <6 days or <7 days into a subject.

[0391] In some embodiments, an amount of modified effector cells is administered to a subject in need thereof and the amount is determined based on the efficacy and the potential of inducing a cytokine-associated toxicity. In another embodiment, the modified effector cells are CAR+ or TCR+ and CD3+ cells. In some cases, an amount of modified effector cells comprises about 10 4 to about 109 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 104 to about 10 5 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 10 5 to about 106 modified effector cells/kg. In some cases, an amount of modified effector cells comprises about 106 to about 10 7 modified effector cells/kg. In some cases, an amount of modified effector cells comprises >10 4 but < 10 5 modified effector cells/kg. In some cases, an amount of modified effector cells comprises >10 5 but < 106 modified effector cells/kg. In some cases, an amount of modified effector cells comprises >106 but < 107 modified effector cells/kg.

[0392] In other embodiments, a method of stimulating the proliferation and/or survival of engineered cells comprises obtaining a sample of cells from a subject, and transfecting cells of the sample of cells with one or more polynucleotides that comprise one or more transposons. In an embodiment, the transposons encode a chimeric antigen receptor (CAR) or a TCR, a cytokine, one or more cell tags, gene switch polypeptides for ligand-inducible control of the cytokine and a transposase effective to integrate said one or more polynucleotides into the genome of said cells, to provide a population of engineered cells. In an embodiment, the gene switch polypeptides comprise i) a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain. In some embodiments, the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In one instance, lymphodepletion is not required prior to administration of the engineered cells to a subject.

[0393] In one instance, a method of in vivo propagation of engineered cells comprises obtaining a sample of cells from a subject, and transfecting cells of the sample of cells with one or more polynucleotides that comprise one or more transposons. In an embodiment, the transposons encode a chimeric antigen receptor (CAR) or a TCR, a cytokine, one or more cell tags, gene switch polypeptides for ligand-inducible control of the cytokine and a transposase effective to integrate said one or more polynucleotides into the genome of said cells, to provide a population of engineered cells. In an embodiment, the gene switch polypeptides comprise i) a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain. In some embodiments, the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In one instance, lymphodepletion is not required prior to administration of the engineered cells to a subject.

[0394] In another embodiment, a method of enhancing in vivo persistence of engineered cells in a subject in need thereof comprises obtaining a sample of cells from a subject, and transfecting cells of the sample of cells with one or more polynucleotides that comprise one or more transposons. In some cases, the transposons encode a chimeric antigen receptor (CAR) or a TCR, a cytokine, one or more cell tags, gene switch polypeptides for ligand-inducible control of the cytokine and a transposase effective to integrate the DNA into the genome of said cells, to provide a population of engineered cells. In some cases, the gene switch polypeptides comprise i) a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain, wherein the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In one instance, lymphodepletion is not required prior to administration of the engineered cells to a subject.

[0395] In another embodiment, a method of treating a subject with a solid tumor comprises obtaining a sample of cells from a subject, transfecting cells of the sample with one or more polynucleotides that comprise one or more transposons, and administering the population of engineered cells to the subject. In one instance, lymphodepletion is not required prior to administration of the engineered cells to a subject. In some cases, the one or more transposons encode a chimeric antigen receptor (CAR) or a TCR, a cytokine, one or more cell tags, gene switch polypeptides for ligand-inducible control of the cytokine and a transposase effective to integrate the DNA into the genome of the cells. In some cases, the gene switch polypeptides comprise: i) a first gene switch polypeptide that comprises a DNA binding domain fused to a first nuclear receptor ligand binding domain, and ii) a second gene switch polypeptide that comprises a transactivation domain fused to a second nuclear receptor ligand binding domain, wherein the first gene switch polypeptide and second gene switch polypeptide are connected by a linker. In some cases, the cells are transfected via electroporation. In some cases, the polynucleotides encoding the gene switch polypeptides are modulated by a promoter. In some cases, the promoter is a tissue-specific promoter or an EF1A promoter or functional variant thereof. In some cases, the tissue-specific promoter comprises a T cell specific response element or an NFAT response element. In some cases, the cytokine comprises at least one of L-1, IL-2, IL-15, IL-12, IL-21, a fusion of IL-15, IL-15Ra, or an IL-15 variant. In some cases, the cytokine is in secreted form. In some cases, the cytokine is in membrane-bound form. In some cases, the cells are NK cells, NKT cells, T-cells or T-cell progenitor cells. In some cases, the cells are administered to a subject (e.g. by infusing the subject with the engineered cells). In some cases, the method further comprises administering an effective amount of a ligand (e.g. veledimex) to induce expression of the cytokine. In some cases, the CAR is capable of binding at least one of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-Al, MUC-16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF R2. In some cases, the transposase is salmonid-type Tcl-like transposase. In some cases, the transposase is SB11 or SB1OOx transposase. In some cases, the cell tag comprise at least one of a HERI truncated variant and a CD20 truncated variant.

Pharmaceutical Compositions and Dosage

[0396] In some embodiments, disclosed herein are compositions comprising a polynucleotide or polypeptide disclosed herein for administration in a subject. In some instances, are modified effector cell compositions encoding a polynucleotide or polypeptide disclosed herein, and optionally containing a cytokine and/or an additional therapeutic agent. In some instances, also included herein are vectors encoding gene-switch polypeptides for regulating expression of a chimeric antigen receptor for modification of an effector cell.

[0397] In some instances, pharmaceutical compositions of a modified effector cell or a vector encoding gene-switch polypeptides and a chimeric antigen receptor are formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

[0398] Pharmaceutical compositions are optionally manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0399] In certain embodiments, compositions may also include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0400] In other embodiments, compositions may also include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0401] The pharmaceutical compositions described herein are administered by any suitable administration route, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, or rectal administration routes. In some instances, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, or intracranial) administration.

[0402] The pharmaceutical compositions described herein are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by an individual to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In some embodiments, the pharmaceutical compositions are formulated into capsules. In some embodiments, the pharmaceutical compositions are formulated into solutions (for example, for IV administration). In some cases, the pharmaceutical composition is formulated as an infusion. In some cases, the pharmaceutical composition is formulated as an injection.

[0403] The pharmaceutical solid dosage forms described herein optionally include a compound described herein and one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof.

[0404] In still other aspects, using standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the compositions. In some embodiments, the compositions are formulated into particles (for example for administration by capsule) and some or all of the particles are coated. In some embodiments, the compositions are formulated into particles (for example for administration by capsule) and some or all of the particles are microencapsulated. In some embodiments, the compositions are formulated into particles (for example for administration by capsule) and some or all of the particles are not microencapsulated and are uncoated.

[0405] In certain embodiments, compositions provided herein may also include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0406] "Proliferative disease" as referred to herein means a unifying concept that excessive proliferation of cells and turnover of cellular matrix contribute significantly to the pathogenesis of several diseases, including cancer is presented.

[0407] "Patient" or "subject" as used herein refers to a mammalian subject diagnosed with or suspected of having or developing a physiological condition, for instance a cancer or an autoimmune condition or an infection. In some embodiments, the term "patient" refers to a mammalian subject with a higher than average likelihood of developing cancer. Exemplary patients may be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents and other mammalians that can benefit from the therapies disclosed herein. Exemplary human patients can be male and/or female.

[0408] "Patient in need thereof' or "subject in need thereof' is referred to herein as a patient diagnosed with or suspected of having a disease or disorder, for instance, but not restricted to a proliferative disorder such as cancer. In some cases, a cancer is a solid tumor or a hematologic malignancy. In some instances, the cancer is a solid tumor. In other instances, the cancer is a hematologic malignancy. In some cases, the cancer is a metastatic cancer. In some cases, the cancer is a relapsed or refractory cancer. In some instances, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, anal cancer; appendix cancer; bile duct cancer (i.e., cholangiocarcinoma); bladder cancer; brain tumor; breast cancer; cervical cancer; colon cancer; cancer of Unknown Primary (CUP); esophageal cancer; eye cancer; fallopian tube cancer; gastroenterological cancer; kidney cancer; liver cancer; lung cancer; medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreatic cancer; parathyroid disease; penile cancer; pituitary tumor; prostate cancer; rectal cancer; skin cancer; stomach cancer; testicular cancer; throat cancer; thyroid cancer; uterine cancer; vaginal cancer; vulvar cancer; or glioblastoma. In some embodiments leukemia can be, for instance, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML).

[0409] "Administering" is referred to herein as providing the compositions of the present disclosure to a patient. By way of example and not limitation, composition administration, e.g., injection, may be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes may be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route. Additionally, administration may also be by surgical deposition of a bolus or pellet of cells, or positioning of a medical device. In an embodiment, a composition of the present disclosure may comprise engineered cells or host cells expressing nucleic acid sequences described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount that is effective to treat or prevent proliferative disorders. A pharmaceutical composition may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0410] As used herein, the terms "treatment," "treating," and its grammatical equivalents refer to obtaining a desired pharmacologic and/or physiologic effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease. To this end, the method described herein comprises administering a "therapeutically effective amount" of the composition comprising the host cells expressing the nucleic acid sequence described herein, or a vector comprising the nucleic acid sequences described herein.

[0411] The terms "therapeutically effective amount", "therapeutic amount", "immunologically effective amount", anti-tumor effective amount", "tumor inhibiting effective amount" or their grammatical equivalents refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of a composition described herein to elicit a desired response in the individual. The precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0412] Alternatively, the pharmacologic and/or physiologic effect of administration of one or more compositions described herein to a patient or a subject may be "prophylactic," i.e., the effect completely or partially prevents a disease or symptom thereof

[0413] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset).

[0414] "Antifoaming agents" reduce foaming during processing which can result in coagulation of aqueous dispersions, bubbles in the finished film, or generally impair processing. Exemplary anti-foaming agents include silicon emulsions or sorbitan sesquoleate.

[0415] "Antioxidants" include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. In certain embodiments, antioxidants enhance chemical stability where required.

[0416] Formulations described herein may benefit from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0417] "Binders" impart cohesive qualities and include, e.g., alginic acid and salts thereof, cellulose derivatives such as carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel@), ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Avicel); microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acids; bentonites; gelatin; polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; tragacanth, dextrin, a sugar, such as sucrose (e.g., Dipac), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab@), and lactose; a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone® XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodium alginate, and the like.

[0418] A "carrier" or "carrier materials" include any commonly used excipients in pharmaceutics and should be selected on the basis of compatibility with compounds disclosed herein, such as, compounds of ibrutinib and An anticancer agent, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. "Pharmaceutically compatible carrier materials" may include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams& Wilkins1999).

[0419] "Dispersing agents," and/or "viscosity modulating agents" include materials that control the diffusion and homogeneity of a drug through liquid media or a granulation method or blend method. In some embodiments, these agents also facilitate the effectiveness of a coating or eroding matrix. Exemplary diffusion facilitators/dispersing agents include, e.g., hydrophilic polymers, electrolytes, Tween @ 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone@), and the carbohydrate-based dispersing agents such as, for example, hydroxypropyl celluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC KI5M, and HPMC K100M), carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl) phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68, F88®, and F108@, which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908@, also known as Poloxamine 908@, which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, N.J.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate copolymer (S-630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates, chitosans and combinations thereof. Plasticizers such as cellulose or triethyl cellulose can also be used as dispersing agents. Dispersing agents particularly useful in liposomal dispersions and self-emulsifying dispersions are dimyristoyl phosphatidyl choline, natural phosphatidyl choline from eggs, natural phosphatidyl glycerol from eggs, cholesterol and isopropyl myristate.

[0420] Combinations of one or more erosion facilitator with one or more diffusion facilitator can also be used in the present compositions.

[0421] The term "diluent" refers to chemical compounds that are used to dilute the compound of interest prior to delivery. Diluents can also be used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain embodiments, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as Di-Pac® (Amstar); mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0422] "Filling agents" include compounds such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0423] "Lubricants" and "glidants" are compounds that prevent, reduce or inhibit adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as CarbowaxTM, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as SyloidTM, Cab-O-Sil@, a starch such as corn starch, silicone oil, a surfactant, and the like.

[0424] "Plasticizers" are compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. In some embodiments, plasticizers can also function as dispersing agents or wetting agents.

[0425] "Solubilizers" include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.

[0426] "Stabilizers" include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.

[0427] "Suspending agents" include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[0428] "Surfactants" include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol

10, octoxynol 40. In some embodiments, surfactants may be included to enhance physical stability or for other purposes.

[0429] "Viscosity enhancing agents" include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

[0430] "Wetting agents" include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

Kits/Article of Manufacture

[0431] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[0432] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[0433] For example, the container(s) include CAR-T cells (e.g., CD19-specific CAR-T cells) encoding gene-switch polypeptides for regulated expression of CARs described herein, and optionally in addition with cytokines and/or chemotherapeutic agents disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

[0434] A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0435] In some embodiments, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.

EXAMPLES

[0436] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1. Nucleofection of PBMC with SB Gene Switch System

[0437] Various DNA plasmids expressing a SB transposon system, i.e. SB11, membrane bound IL-15 (mbIL-15), and chimeric antigen receptor (CAR), were transfected to peripheral blood mononuclear cells (PBMC) via nucleofection to redirect T cell specificity (Figure 1).

[0438] On day 0, 20 million PBMC were resuspended in 100 pL of Amaxa Human T cell Nucleofector solution (Cat. no. VPA-1002; Lonza, Basel, Switzerland) mixed with a total of 15 ptg of transposons and 5 pg of transposase (SB11) and electroporated.

[0439] The following day (day 1) cells were counted, and CAR expression was measured by flow cytometry. CAR T Cells were stimulated with either y-irradiated (100 Gy) or mitomycin C treated AaPCs ata 1:1 ratio. The AaPC cells used were K562-AaPC expressing CD19 antigen. Cultures were supplemented with IL-21 (30ng/ml) only for the first round of stimulation and subsequently with recombinant human TL-2 (50 IU/ml) and IL-21 (30 ng/ml) (Pepro Tech) for remaining stimulations. T cell cultures were phenotyped at the end of each stimulation cycle, which typically lasted 7 days. The cultures were phenotyped for CAR expression by multi parameter flow cytometry utilizing either Protein L or anti-idiotype antibody that recognizes CD19-specificCAR. Cultures were also closely monitored for the outgrowth of NK cells (defined as CD3negCD56+ population) and were removed from the CAR T cell cultures when the percentage exceeded 10% of total cell populations using magnetic beads for specific for CD56 (Stem Cell Technologies and/or Miltenyi Biotec), according to the manufacturer's instructions.

Example 2. Flow Cytometry Analysis of ON-OFF SB Gene Switch System

[0440] CD19-specific T cells were generated from mononuclear cells (MNC) derived from PB or UCB using SB transposition to introduce the CAR followed by addition of AaPC to numerically expand the T cells in a CAR-dependent manner.

[0441] For flow cytometry analysis to assess expression of various markers and gene(s) of interest, cells were gently resuspended and cell number, and viability were measured using

Trypan blue exclusion method with the Countess instrument. Cell diameter size was also recorded. 5 x 105 cells for each for the samples were harvested at 330xg for 4 min at 10°C for antibody staining. Harvested cells were incubated on ice for at least 15 min with 10% human AB serum in HBSS. Antibody cocktails containing fluorescently conjugated antibodies included one or more of antibodies specific to CD4 (Clone RPA-T4), CD8 (Clone SKI), CD3 (Clone UCHT1), CD56, CD19-specific CAR (anti-idiotype antibody), IL-15, and IL-15Ra, in HBSS+0.1%BSA+2mMEDTA. The prepared antibody cocktails and associated fluorescence minus one/isotype control were added to stain the cell samples, and then the samples were incubated on ice for 30 min. The samples were then washed then with FACS buffer (HBSS

+ 0.5% BSA + 2mM EDTA) and stained with Fixable Viability Dye (eBiosciences) for 30 min on ice. Cells were washed with FACS buffer and then fixed with a 4% paraformaldehyde solution (BD Cytofix; BD Biosciences). All samples were run on a LSR II flow cytometer, a Fortessa X 20 flow cytometer (BD Biosciences) or iQue Screener Plus (Intellicyt) and data was analyzed using FlowJo V10 (TreeStar, Ashland, OR) or iQue Screener software.

[0442] For induction of expression using ligand (veledimex), AaPC stimulated CAR-T cells were harvested at 1x 10 cells/ml. Either veledimex or DMSO (to keep DMSO concentration constant in both cultures) was added to the cultures. Veledimex was used at a concentration of 20nM, 50nM, 100nM or as described. Cells were then cultured at 37°C in presence of veledimex or DMSO for 2-3 days. After 2-3 days in culture, 5 x10 5 cells for each for the samples were harvested at 330xg for 4 min at 10°C for antibody staining. Harvested cells were incubated on ice for at least 15 min with 10% human AB serum in HBSS. Antibody cocktails containing fluorescently conjugated antibodies included one of more of antibodies specific to CD4 (Clone RPA-T4), CD8 (Clone SKI), CD3 (Clone UCHT1), CD56, CD19-specific CAR (anti-idiotype antibody), IL-15, and IL-15Ra inTHBSS+0.1% BSA+2mMEDTA. The prepared antibody cocktails and associated isotype control were added to stain the cell samples, and then the samples were incubated on ice for 30 min. The samples were then washed with FACS buffer (HBSS + 0.5% BSA + 2mM EDTA) and stained with Fixable Viability Dye (eBiosciences) for 30 min on ice. Cells were washed with FACS buffer and fixed with 4% paraformaldehyde solution (BD Cytofix; BD Biosciences). All samples were run on a LSR II flow cytometer, a Fortessa X-20 flow cytometer (BD Biosciences) or iQue Screener Plus (Intellicyt) and data was analyzed using FlowJo V1O (TreeStar, Ashland, OR) or iQue Screener software.

[0443] For veledimex ON-OFF-ON experiments, veledimex was first added at mentioned concentration (typically between 20-100 nM) to cell cultures for 2-3 days. An aliquot of cells was harvested and analyzed for expression (ON) of various proteins by flow cytometry as noted above. Remaining cell culture was then washed and resuspended in complete medium without veledimex and cultured for up to 5 days. Aliquots of cells were harvested and analyzed for expression (OFF) at times mentioned by flow cytometry as noted above. Remaining cell culture was then washed and resuspended with complete medium containing veledimex to turn the expression of target gene(s) back on. After 2-3 days in culture, aliquot of cells were harvested and analyzed for expression by flow cytometry.

Example 3. Survival Experiments

[0444] Multiple Sleeping Beauty transposon DNA plasmids encoding for RTS-membrane bound IL-15 (RTS-mbIL-15), CD19-specific chimeric antigen receptor (CD19 CAR) (Design 9, Fig. 2) were transfected into peripheral blood mononuclear cells (PBMC) along with plasmid DNA encoding for Sleeping Beauty transposase SB11 via nucleofection (Figure 1). On day 0, 20 million PBMC were resuspended in 100 pL of Amaxa Human T cell Nucleofector solution (Cat. no. VPA-1002; Lonza, Basel, Switzerland) mixed with a total of 15 pg of transposons and 5 ptg of transposase and nucleofected into cells. The following day (dayl) cells were counted, and CAR expression was measured using flow cytometry. Cells were stimulated with either y irradiated (100 Gy) or mitomycin C treated K562 based AaPCs expressing CD19 on their surface at a 1:1 ratio. Cultures were supplemented with IL-21 (30ng/ml) only for the first round of stimulation and subsequently with recombinant human TL-2 (50 IU/ml) and IL-21 (30 ng/ml) (Pepro Tech) for remaining stimulations. T cell cultures were phenotyped at the end of each stimulation cycle, which typically lasted 7 days. The cultures were phenotyped for CAR expression, utilizing anti-idiotype antibody staining to detect CD19-specific CAR-T cells by multi-parameter flow cytometry. Cultures were also closely monitored for the outgrowth of NK cells (defined as CD3negCD56+ population) and were removed from the CAR T cell cultures when the percentage exceeded 10% of total cell populations using magnetic beads for CD56 (Stem Cell Technologies and/or Miltenyi Biotec), according to the manufacturer's instructions.

[0445] To measure persistence of CD19-specific CAR-T cells co-expressingRTS-mbIL-15 upon withdrawal of cytokines (IL-2 and IL-21) from culture media, CAR-T cells were harvested and seeded in complete RPMI medium with or without IL-2 and IL-21, at the concentration of 1x106 cells/ml in 6-well plates. Veledimex (50 nM final concentration) or DMSO control were added to the culture media. Culture medium was changed every 2-3 days and small aliquot of cells were harvested for flow analysis. Flow analysis was conducted to measure mbIL-15 expression as well as to measure the % of live cells in the culture in presence of absence of cytokines from media. Survival of CAR-T cells is shown in Figure 12 in presence and absence of cytokines. In absence of cytokines in culture, improved survival of CAR-T cells was seen when veledimex was added to induce mbIL-15 expression. When veledimex was not added (mbIL-15 expression was not induced), cells did not survive in ex vivo culture.

Example 4. Western Blotting

[0446] PBMC were nucleofected with plasmids of Sleeping Beauty system to express CD19 specific CAR under constitutive promoter (CD19 CAR alone), or CD19-specific CAR and mbIL 15 both under constitutive promoters (CD19 CAR + constitutive-mbL-15) or CD19-specific CAR under constitutive promoter and RTS-mbIL-15 (CD19 CAR + RTS-mbIL-15). Nucleofected cells were cultured in presence of AaPC as described in Example 1.

[0447] After four rounds of stimulations, cells were cultured in absence (CD19 CAR alone, CD19 CAR + constitutive-mbIL-15 and CD19 CAR + RTS-mbIL-15) or presence (CD19 CAR

+ RTS-mbIL-15) of veledimex for 2-3 days. Cell lysates were prepared for western blot analysis. Approximately 10pg lysate/lane of NuPAGE 10% Bis-Tris gel was loaded. Proteins were transferred from gel to a polyvinylidene fluoride (PVDF) membranes using the iBlot@ (Life Technologies) semi-dry transfer apparatus. Membrane was blocked using a 5% (w/v) powdered milk solution in a PBS+Tween-20 (PBST; 1X PBS + 0.05% Tween-20) solution stained with goat anti-human IL-15 (R&D Systems) primary antibody and rabbit anti-goat IgGHIRP (KPL Laboratories) secondary antibody. SuperSignalTMWest Pico Chemiluminescent Substrate (Thermo Fisher Scientific) for enhanced chemiluminescence (ECL) detection was utilized.

[0448] Image of the western blot were captured on the FluorChemTM E Imager (ProteinSimple, San Jose, CA) system using the Digital Darkroom software and AlphaView@ software (ProteinSimple). Figure 14 shows image of western blot. mbIL-15 expression was not detected by western blot when veledimex was absent (CD19 CAR + RTS-mbIL-15), but strong expression of mbIL-15 was observed when veledimex was added to the culture. InCD19CARonly negative control, no mbIL-15 expression was observed. In CD19 CAR + constitutive-mbIL-15 positive control, expression of mbL-15 was detected.

Example 5. T Cell Activation and Ligand Specific Control of mbIL-15 Transgene Expression

[0449] Sleeping Beauty transposons were designed to stably express CD19-specific CAR, a cell tag as well as gene switch- controlled mbIL-15. Expression of one or both of gene switch components (e.g. VP16/RxR and Gal4/EcR) were performed under the control of a T cell activation specific promoter. In this example, a T cell activation dependent promoter generated by 6X NFAT response element fused to minimal IL-2 promoter (NFAT6-IL2 promoter) was utilized to drive expression of gene switch components- VP16-RxR and/or Gal4-EcR fusion proteins for conditional expression of mbIL-15. When components of the gene switch are under the control of NFAT6-IL2 promoter, expression of mbIL-15 requires two conditions to be met: 1) T cells are activated ; and 2) activator ligand, veledimex, to be present. SB transposons construct combinations shown in Fig. 20A were transfected along with SB11 transposase in donor T cells.

[0450] On day 0, 10-20 million T cells were resuspended in 100 pL of Amaxa Human T cell Nucleofector solution (Cat. no. VPA-1002; Lonza, Basel, Switzerland) mixed with a total of 15 ptg of transposon(s) and 5 pg of transposase (SB11) and electroporated.

[0451] The following day (day 1) cells were counted, and CAR expression was measured by flow cytometry. CAR-T Cells were selectively expanded ex vivo by successive rounds of stimulations every 7-10 days for up to 4 cycles with either y-irradiated (100 Gy) or mitomycin C treated AaPCs ata 1:1 ratio. The AaPC cells used were K562-AaPC expressing CD19 antigen. Cultures were supplemented with IL-21 (30ng/ml) only for the first round of stimulation and subsequently with recombinant human L-2 (50 IU/ml) and IL-21 (30 ng/ml) (Pepro Tech) for remaining stimulations.

[0452] For flow cytometry analysis to assess expression of various markers and gene(s) of interest cells were gently resuspended and cell number and viability were measured using Trypan blue exclusion method with the Countess instrument. Cell diameter size was also recorded. 5x10 5 cells for each for the samples were harvested at 330xg for 4 min at 10°C for antibody staining. Harvested cells were incubated on ice for at least 15 min with 10% human AB serum in HBSS. Antibody cocktails containing fluorescently conjugated antibodies included one or more of antibodies specific to CD4, CD8, CD3, CD56, CD19-specific CAR (anti-idiotype antibody), IL-15 and/or IL-15Ra, inTHBSS+0.1% BSA+2mMEDTA. The prepared antibody cocktails and associated fluorescence minus one/isotype control were added to stain the cell samples, and then the samples were incubated on ice for 30 min. The samples were then washed then with FACS buffer (HBSS + 0.5% BSA + 2mM EDTA) and stained with Fixable Viability Dye (eBiosciences) for 30 min on ice. Cells were washed with FACS buffer and then fixed with a 4% paraformaldehyde solution (BD Cytofix; BD Biosciences). All samples were run on a LSR II flow cytometer, a Fortessa X-20 flow cytometer (BD Biosciences) or iQue Screener Plus (Intellicyt) and data was analyzed using FlowJo V1O (TreeStar, Ashland, OR) or iQue Screener software.

[0453] To assess the impact of T cell activation on mbIL-15 expression, cells were kept in culture media for at least 7 days post last AaPC stimulation to test baseline (no stimulation) expression levels. T cells were activated using different doses of ConA (1 or 5 pg/mL) for 48 hours or PMA/Ionomycin for 24 hours prior to treatment with veledimex or DMSO.

[0454] Non-activated or activated CAR-T cells were harvested and either 100nMveledimex or DMSO (to keep DMSO concentration constant in both cultures) was added to the cultures. Cells were then cultured at 37C in presence of veledimex or DMSO for at least 24 hours before flow cytometry analysis to measure transgene expression. CAR and mbIL-15 expression was quantified in either resting (Fig. 20B) or activated T cells in presence of absence of veledimex (Fig. 20C: ConA 1 g/mL; ConA 5 pg/mL: Fig. 20D, PMA/Ionomycin: Fig. 20E).

[0455] Fig. 20B shows that in absence of T cell activation, only CAR-T cells that harbor transposons for expression of gene switch components (e.g. VP16/RxR and Gal4/EcR) under a constitutive promoter were able to express mbIL-15 in when veledimex was added to the culture. As shown in Figs. 20C through 20E, CAR-T cells harboring transposons for expression of one or more gene switch components under NFAT6-IL-2 promoter (constructs #3, 4 and 6) exhibited expression of mbIL-15 when veledimex was added to culture showing two conditions had to be met for inducible expression of mbIL-15 by CAR-T cells. Furthermore, levels of mbIL-15 expression in presence of veledimex at fixed concentration increased with level of T cells activation (Fig. 20C and Fig. 20D). Extremely low levels of mbIL-15 expression by flow cytometry were detected in absence of veledimex for all tested construct combinations. Consistent with the design of these ligand inducible switch systems, only CAR+ T cells were able to induce mbIL-15 in presence of veledimex.

Example 6. Expression of IL-12 Using Gene Switch System

[0456] Sleeping Beauty transposons were designed as depicted in Fig. 22 to stably express EGFRvIII-specific CAR, a cell tag and RTS-IL-12. Transposon plasmids were electroporated in several donor T cells as previously described. EGFRvIII-CAR-T cells were selectively expanded ex vivo by successive rounds of stimulations every 7-10 days for up to 4 cycles with either y irradiated or mitomycin C treated AaPC that served as "feeder cells" at 1:1 ratio in media supplemented with IL-2 and IL-21. The AaPC cells used were K562-AaPC expressing EGFRvIII antigen. Fig. 26 depicts CAR expression in T cells from multiple donors as the CAR+ T cells are selectively expanded ex vivo.

[0457] Veledimex ON-OFF cycling experiments: For each ON-OFF cycle, veledimex was first added (ON) at previously mentioned concentrations (typically between 20-100 nM) to cell cultures for 2-3 days. An aliquot of cells was harvested and analyzed for expression (ON) of various proteins by flow cytometry and/or ELISA at noted time points. Remaining cell culture was then washed and resuspended in complete medium without veledimex (OFF) and cultured for up to 5 days. Aliquots of cells were harvested and analyzed for expression (OFF) at times mentioned by flow cytometry and/or ELISA as noted above. Veledimex ON-OFF cycle(s) can be repeated again with remaining cell culture by repeating the process.

[0458] To assay IL-12 induction in EGFRvIII-CAR-T cells, Pan T-cells at 24h post nucleofection were treated with DMSO (control) or veledemix in the presence of AaPCs in media containing TL-21. After 48 hours post-nucleofection, culture supernatants were analyzed for IL 12 levels by ELISA.

[0459] FIG. 27 shows that treatment with veledimex induced IL-12 expression by T cells that were transfected with SB transposons to co-express both CAR and RTS-IL-12. CAR-T cells lacking RTS-IL-12 however, failed to produce IL-12 in culture supernatant. Treatment with DMSO control showed low background levels of IL-12 induction.

[0460] Furthermore, the ability of RTS-IL-12/CAR-T cells to produce inducible IL-12 was assessed in presence or absence of antigen specific stimulation. EGFRvIII-CAR/RTS-IL-12 T cells were expanded ex vivo by three successive rounds of stimulation with EGFRvIII AaPCs. Veledimex (100 nM) or DMSO (control) were added to the culture of EGFRvIII-CAR/RTS-IL 12 T cells in absence (Fig. 28A) or presence (Fig. 28B) of EGFRvIII AaPCs for 48 hours before analysis of IL-12 levels in culture supernatant. As shown in Fig. 28A and Fig. 28B, the ability of EGFRvIII-CAR/RTS-IL-12 T cells to express veledimex induced IL-12 is greatly enhanced in presence of EGFRvIII antigen specific stimulation with low background levels of IL-12 expression in absence of veledimex.

Example 7. Veledimex ON-OFF Cycling in EGFRvIII CAR Expressing Cells Using RTS Gene Switch System

[0461] The effect of repeated treatments and withdrawals of veledimex to EGFRvIII-CAR-T cells harboring an inducible IL-12 gene was determined in vitro both in the presence and absence of antigen specific T cell activation by co-culture with AaPCs. In the first cycle (ON-OFF) of ligand treatment, CAR-T cells (10 6/ml) expanded ex vivo by four successive rounds of stimulation with EGFRvIIIF AaPCs were treated with DMSO or veledimex and cultured in the presence or absence of AaPCs in media containing IL-21 and IL-2. After 48 hours post treatment, cells were washed thoroughly to remove ligand and cultured in a new culture vessel in media containing IL-21 and IL-2 for 5 days. Culture supernatants harvested between 24-120 hours post-treatment were then analyzed for expression of IL-12 and IFN.

[0462] In the second cycle (ON-OFF) of ligand treatment, CAR-T cells from cultures treated with DMSO or veledimex in the presence of AaPCs in cycle 1 were collected at 5 days after withdrawal of veledimex (in cycle 1) and subjected to a second cycle of ligand treatment with DMSO or veledimex in the presence or absence of antigen specific T cell activation by co culture with AaPCs. After 48 hours post-treatment, cells were washed thoroughly to remove ligand and cultured in a new culture vessel in media containing IL-21 plus IL-2 for 5 days. Culture supernatants harvested between 24-120 hours post-treatment were then analyzed for expression of IL-12 and IFNy.

[0463] FIG. 29A shows that treatment with veledimex in EGFRvIII-CAR-T cells also harboring inducible IL-12 constructs (XON-61 or XON-62; SEQ ID NO 135 or 136) results in an increase in IL-12 expression relative to treatment with DMSO in culture supernatants at 48h post treatment. Levels of IL-12 induction were enhanced in the presence of AaPC-mediated antigen specific stimulation compared to the absence of antigen-specific stimulation. Very low background levels of IL-12 expression were observed in absence of veledimex in presence or absence of antigen specific stimulation. EGFRvIII-CAR-T cells without inducible IL-12 failed to express any IL-12 in presence or absence of antigen specific stimulation.

[0464] The effect of withdrawing veledimex on IL-12 expression is shown in FIG. 30A (cells cultured in the absence of AaPCs) and FIG. 30B (cells cultured in the presence of AaPCs). In both the presence and absence of AaPC-mediated antigen-specific stimulation, withdrawal of veledimex at 48h-post-treatment resulted in a reduction of IL-12 expression. Levels of IL-12 induction were significantly lower in the absence of antigen-specific stimulation (FIG. 30A) compared to in the presence of antigen-specific stimulation (FIG. 30B). But levels of IL-12 gradually reduced back to the baseline levels upon withdrawal of veledimex exhibiting tight control of inducible gene expression.

[0465] FIG. 31A and FIG. 31B show levels of IL-12 induction in culture supernatants at48h post-ligand treatment during a second cycle with veledimex or DMSO following withdrawal of the agents during cycle 1. DMSO or veledimex treated cells cultured in the presence of EGFRvIII-expressing AaPCs in cycle 1were treated with veledimex (or DMSO) in cycle 2 in the presence or absence of EGFRvIII~aAaPCs. Results show that cells treated with veledimex and EGFRvIIIW AaPCs in cycle 2 show increased IL-12 induction when they were treated with veledimex and EGFRvIII- AaPCs during cycle 1 (FIG. 31B) relative to cells treated with DMSO and EGFRvIII AaPCs during cycle 1 (FIG. 31A). Again very low background levels of IL-12 expression were observed in absence of veledimex.

[0466] Kinetics of RTS- IL-12 expression upon two cycles of treatment with veledimex is captured in FIG. 32A. EGFRvIII-CAR-T cells harboring inducible IL-12 constructs (XON-61 or XON-62; SEQ ID NO 135 or 136) were evaluated for their ability to induce IL-12 expression in presence of absence of veledimex by two cycles of ligand treatment as described above. Ligand inducible gene switch vectors in this example are designed such that only cells that express both transposons are capable of gene switch controlled expression of IL-12. Expression of EGFRvIII

CAR was quantified by flow cytometry in T cells prior to start of cycle 1 of ligand treatment as shown in Fig. 34B. Cells were gated on CD3.

[0467] As shown in FIG. 32A, IL-12 expression by these CAR-T cells can be turned ON and OFF repeatedly by addition or withdrawal of activator ligand veledimex during prolonged culture period. IL-12 expression went down to baseline levels upon withdrawal of veledimex and can be turned back on upon addition of veledimex.The data is representative from one donor.

[0468] FIG. 33 shows another example of kinetics of RTS-IL-12 expression upon two cycles of treatment with veledimex. EGFRvIII-CAR-T cells harboring inducible IL-12 constructs (XON-61 or XON-62; SEQ ID NO 135 or 136) were evaluated for their ability to induce IL-12 expression in presence of absence of veledimex by two cycles of ligand treatment as described above. In this case, the first treatment cycle was carried out in the presence of EGFRvIII AaPCs and the second cycle was in the absence of EGFRvIIIWAaPCs. Consistent with previous finding, veledimex can regulate IL-12 expression by EGFRvIII-CAR-T cells upon repeated treatment and withdrawal during prolonged culture period. Removal of AaPC-mediated EGFRvIII-specific stimulation during cycle 2 leads to a corresponding reduction of IL-12 levels. The data is representative from a second donor.

Example 8. Veledimex dose-response

[0469] EGFRvIII-CAR T cells with or without RTS-IL-12 were expanded ex vivo by three successive rounds of stimulation with EGFRvIIIF AaPCs. T cells were then treated with varying doses of veledimex from 0 nM (DMSO control) to 100 nM. At 48h post veledimex treatment, culture supernatants were harvested analyzed for IL-12 levels by ELISA. EGFRvIII-CAR expression in all groups was confirmed with flow cytometry and was stable with increasing doses of veledimex (data not shown) as expected with CAR expression under control of a constitutive promoter.

[0470] Results are shown in FIG. 34. Results show that, EGFRvIII-CAR/RTS-IL-12 T cells produced IL-12 in veledimex dose dependent manner. Lower levels of IL-12 expression was observed for a given veledimex concentration in cells transfected with transposons that express a cell tag in addition to CAR and IL-12 compared to cells lacking cell tag expression. EGFRvIII CAR-T cells without RTS-IL-12 failed to express IL-12.

Example 9. Specific cytotoxicity of EGFRvIII-CAR-T cells towards EGFRvIII target cells

[0471] The ability of T-cells expressing EGFRvIII-CARto respond to EGFRvIII+ target cells was tested. EGFRvIII-CAR T cells with or without RTS-IL-12 were expanded ex vivo by four successive rounds of stimulation with EGFRvIII AaPCs. These effector EGFRvIII-CAR-T cells were rested for 48h in RPMI containing 2% FBS and L-glutamine prior to co-culture with and E andGFRvIIIe target cells at a 1:1 effector to target cell ratio in absence of veledimex. Culture supernatants were collected at 24h post-culture and analyzed for IFNy as a functional readout of specificity of EGFRvIII-CAR-T cells.

[0472] FIG. 35 shows EGFRvIII antigen specific IFNy production by CAR-T cells at 24h post co-culture with target cells. Co-culture with EGFRvIII'1EL4 cell line or effector CAR-T cells alone did not result in IFNy expression.

[0473] FIG. 36A shows IFNy in culture supernatants at 24h post co-culture with EGFRvIII expressing Glioblastoma target cells (no veledimex). Results show that EGFRvIII Glioblastoma target cells (U251MG-EGFRvIII-Fluc-GFP and U87MG-EGFRvIII-Fluc-GFP) induced IFNy secretion by EGFRvIII-CAR T-cells while EGFRvIII'1 target cells (U251MG, U251MG EGFRvIII-Fluc-GFP, U87MG, and U87MG-Fluc-GFP) or effector cells alone failed to induce IFNy secretion.

[0474] Cytotoxicity of EGFRvII-CAR-T cells towards EGFRvIII and EGFRvIII target cells was tested in a 2 hr Europium release assay. Target cell lines were labeled using the DELFIA BATDA reagent (DELFIA EuTDA Cytotoxicity assay; Perkin Elmer). EGFRvIII-CAR-T effector (E) cells were co-cultured with labeled target (T) cells at (E:T) ratios of 3:1, 1:1 or 0.3:1 in RPMI-1640 + 5% heat-inactivated FBS. After 2 hr, supernatant from the co-cultures were harvested and developed with addition of the DELFIA Europium assay and read on a time resolved fluorescence instrument to measure cytotoxicity of target cells. The results from example experiments are depicted in Fig. 36B.

[0475] As shown in Fig. 36B, EGFRvIII-CAR-T cells with or without RTS-IL-12 showed EGFRvIII antigen specific, dose-dependent cytotoxicity of target cells.

Example 10. Nucleofection of PBMCs with Serine Recombinase System

[0476] Various DNA plasmids containing serine recombinase attachment sites and components of the gene switch system as depicted in Figure 17 were transfected to peripheral blood mononuclear cells (PBMC) via nucleofection to redirect T cell specificity. Specifically, 3 single vectors were tested in the presence and absence of a serine recombinase, SF370: (i) CAR:HERlt +constitutive mbIL15 (positive control) (ii) CAR:HBERlt+ promoterless mbIL 15 (negative control) (iii) CAR:HERlt+RTS:mbI15 The expression of the CAR and mbIL15 was evaluated weekly (at days 1, day 8, day 15 and day 22). Aliquots of cells were treated with either DMSO or veledimex 24-48h prior to each flow to assess inducible mbIL15 expression. AaPC Clone 1 was added weekly at a 1:1 ratio with CAR+ cells.

[0477] Only cells receiving the CAR/mbIL15 vector construct + SF370 recombinase were observed to expand; all other cultures failed to expand within a week despite similar transfection efficiencies. This result is indicative of recombinase-mediated integration of the test construct. HERlt expression was only evaluated on days 22 & 29 and correlates well with CAR expression (data not shown).

[0478] Two weeks post-transfection, veledimex treatment (-48h) was associated with an induction of mbIL-15 expression. In the absence of veledimex, mbIL-15 expression is in the range with the negative control "promoterless mbIL15" sample. Removal of veledimex was associated with complete loss of mbIL15 expression.

[0479] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments described herein, or combinations of one or more of these embodiments or aspects described therein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0480] Any reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.

50471‐706_601_SL.TXT 50471-706_601_SL.TXT SEQUENCE LISTING SEQUENCE LISTING

<110> INTREXON CORPORATION <110> INTREXON CORPORATION <120> MODULATING EXPRESSION OF POLYPEPTIDES VIA NEW GENE SWITCH <120> MODULATING EXPRESSION OF POLYPEPTIDES VIA NEW GENE SWITCH EXPRESSION SYSTEMS EXPRESSION SYSTEMS

<130> 50471‐706.601 <130> 50471-706.601

<140> <140> <141> <141>

<150> 62/464,958 <150> 62/464,958 <151> 2017‐02‐28 <151> 2017-02-28

<150> 62/444,775 <150> 62/444,775 <151> 2017‐01‐10 <151> 2017-01-10

<160> 244 <160> 244

<170> PatentIn version 3.5 <170> PatentIn version 3.5

<210> 1 <210> 1 <211> 54 <211> 54 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" oligonucleotide'

<220> <220> <221> source <221> source <223> /note="Whitlow Linker" <223> /note="Whitlow Linker"

<400> 1 <400> 1 ggcagcacct ccggcagcgg caagcctggc agcggcgagg gcagcaccaa gggc 54 ggcagcacct ccggcagcgg caagcctggc agcggcgagg gcagcaccaa gggc 54

<210> 2 <210> 2 <211> 78 <211> 78 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" oligonucleotide"

<220> <220> <221> source <221> source Page 1 Page 1

50471‐706_601_SL.TXT 50471-706_601_SL.TXT - <223> /note="Linker" <223> /note="Linker"

<400> 2 <400> 2 tctggcggag gatctggagg aggcggatct ggaggaggag gcagtggagg cggaggatct 60 tctggcggag gatctggagg aggcggatct ggaggaggag gcagtggagg cggaggatct 60

ggcggaggat ctctgcag 78 ggcggaggat ctctgcag 78

<210> 3 <210> 3 <211> 9 <211> 9 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="GSG linker" <223> /note="GSG linker"

<400> 3 <400> 3 ggaagcgga 9 ggaagcgga 9

<210> 4 <210> 4 <211> 12 <211> 12 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="SGSG linker" <223> /note="SGSG linker"

<400> 4 <400> 4 agtggcagcg gc 12 agtggcagcg gc 12

<210> 5 <210> 5 <211> 45 <211> 45 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic Page 2 Page 2

50471‐706_601_SL.TXT 50471-706_601_SL.TXT oligonucleotide" oligonucleotide"

<220> <220> <221> source <221> source <223> /note="(G4S)3 linker" <223> /note=" (G4S)3 linker"

<400> 5 <400> 5 ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45 ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45

<210> 6 <210> 6 <211> 12 <211> 12 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Furin cleavage site/ Furinlink1" <223> /note="Furin cleavage site/ Furinlink1"

<400> 6 <400> 6 cgtgcaaagc gt 12 cgtgcaaagc gt 12

<210> 7 <210> 7 <211> 84 <211> 84 <212> DNA <212> DNA <213> Foot‐and‐mouth disease virus <213> Foot-and-mouth disease virus

<220> <220> <221> source <221> source <223> /note="Fmdv" <223> /note="Fmdv"

<400> 7 <400> 7 agagccaaga gggcaccggt gaaacagact ttgaattttg accttctgaa gttggcagga 60 agagccaaga gggcaccggt gaaacagact ttgaattttg accttctgaa gttggcagga 60

gacgttgagt ccaaccctgg gccc 84 gacgttgagt ccaaccctgg gccc 84

<210> 8 <210> 8 <211> 54 <211> 54 <212> DNA <212> DNA <213> Thosea asigna virus <213> Thosea asigna virus

<220> <220> <221> source <221> source <223> /note="Thosea asigna virus 2A region (T2A)" <223> /note="Thosea asigna virus 2A region (T2A)"

Page 3 Page 3

50471‐706_601_SL.TXT 50471-706_601_SL.TXT - <400> 8 <400> 8 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg acct 54 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg acct 54

<210> 9 <210> 9 <211> 75 <211> 75 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Furin‐GSG‐T2A" <223> /note="Furin-GSG-T2A'

<400> 9 <400> 9 agagctaaga ggggaagcgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag 60 agagctaaga ggggaagcgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag 60

gagaatcctg gacct 75 gagaatcctg gacct 75

<210> 10 <210> 10 <211> 78 <211> 78 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Furin‐SGSG‐T2A" <223> /note="Furin-SGSG-T2A'

<400> 10 <400> 10 agggccaaga ggagtggcag cggcgagggc agaggaagtc ttctaacatg cggtgacgtg 60 agggccaaga ggagtggcag cggcgagggc agaggaagto ttctaacatg cggtgacgtg 60

gaggagaatc ccggccct 78 gaggagaatc ccggccct 78

<210> 11 <210> 11 <211> 57 <211> 57 <212> DNA <212> DNA <213> Porcine teschovirus <213> Porcine teschovirus

<220> <220> <221> source <221> source <223> /note="Porcine teschovirus‐1 2A region (P2A)" <223> /note="Porcine teschovirus- 1 2A region (P2A)" "

Page 4 Page 4

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<400> 11 <400> 11 gcaacgaact tctctctcct aaaacaggct ggtgatgtgg aggagaatcc tggtcca 57 gcaacgaact tctctctcct aaaacaggct ggtgatgtgg aggagaatcc tggtcca 57

<210> 12 <210> 12 <211> 66 <211> 66 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="GSG‐P2A" <223> /note="GSG-P2A"

<400> 12 <400> 12 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60

ggacct 66 ggacct 66

<210> 13 <210> 13 <211> 60 <211> 60 <212> DNA <212> DNA <213> Equine rhinitis A virus <213> Equine rhinitis A virus

<220> <220> <221> source <221> source <223> /note="Equine rhinitis A virus 2A region (E2A)" <223> /note="Equine rhinitis A virus 2A region (E2A)" "

<400> 13 <400> 13 cagtgtacta attatgctct cttgaaattg gctggagatg ttgagagcaa ccctggacct 60 cagtgtacta attatgctct cttgaaattg gctggagatg ttgagagcaa ccctggacct 60

<210> 14 <210> 14 <211> 66 <211> 66 <212> DNA <212> DNA <213> Foot‐and‐mouth disease virus <213> Foot-and-mouth disease virus

<220> <220> <221> source <221> source <223> /note="Foot‐and‐mouth disease virus 2A region (F2A)" <223> /note="Foot-and-mouth disease virus 2A region (F2A)" "

<400> 14 <400> 14 gtcaaacaga ccctaaactt tgatctgcta aaactggccg gggatgtgga aagtaatccc 60 gtcaaacaga ccctaaactt tgatctgcta aaactggccg gggatgtgga aagtaatcco 60

ggcccc 66 ggcccc 66

Page 5 Page 5

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<210> 15 <210> 15 <211> 93 <211> 93 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="FP2A" <223> /note="FP2A"

<400> 15 <400> 15 cgtgcaaagc gtgcaccggt gaaacaggga agcggagcta ctaacttcag cctgctgaag 60 cgtgcaaagc gtgcaccggt gaaacaggga agcggagcta ctaacttcag cctgctgaag 60

caggctggag acgtggagga gaaccctgga cct 93 caggctggag acgtggagga gaaccctgga cct 93

<210> 16 <210> 16 <211> 24 <211> 24 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Linker‐GSG" <223> /note="Linker-GSG"

<400> 16 <400> 16 gcaccggtga aacagggaag cgga 24 gcaccggtga aacagggaag cgga 24

<210> 17 <210> 17 <211> 15 <211> 15 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Linker" <223> /note="Linker"

Page 6 Page 6

50471‐706_601_SL.TXT 50471-706_601_SL.TXT -

<400> 17 <400> 17 gcaccggtga aacag 15 gcaccggtga aacag 15

<210> 18 <210> 18 <211> 570 <211> 570 <212> DNA <212> DNA <213> Encephalomyocarditis virus <213> Encephalomyocarditis virus

<220> <220> <221> source <221> source <223> /note="EMCV IRES" <223> /note="EMCV IRES"

<400> 18 <400> 18 ccccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60 ccccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240 gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagad 240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg 540 gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg 540

ggacgtggtt ttcctttgaa aaacacgatc 570 ggacgtggtt ttcctttgaa aaacacgato 570

<210> 19 <210> 19 <211> 73 <211> 73 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="2xRbm3 IRES" <223> /note="2xRbm3 IRES"

<400> 19 <400> 19 actagtttta taatttcttc ttccagaatt tctgacattt tataatttct tcttccagaa 60 actagtttta taatttcttc ttccagaatt tctgacattt tataatttct tcttccagaa 60 Page 7 Page 7

50471‐706_601_SL.TXT 50471-706_601_SL.TXT -

gactcacaac ctc 73 gactcacaac ctc 73

<210> 20 <210> 20 <211> 66 <211> 66 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="GM‐CSFR‐alpha signal peptide" <223> /note="GM-CSFR-alpha signal peptide"

<400> 20 <400> 20 atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60 atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccago attcctcctg 60

atccca 66 atccca 66

<210> 21 <210> 21 <211> 66 <211> 66 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<400> 21 <400> 21 atgctgctgc tggtgaccag cctgctgctg tgtgagctgc cccaccccgc ctttctgctg 60 atgctgctgc tggtgaccag cctgctgctg tgtgagctgc cccaccccgc ctttctgctg 60

atcccc 66 atcccc 66

<210> 22 <210> 22 <211> 60 <211> 60 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic Page 8 Page 8

50471‐706_601_SL.TXT 50471-706_601_SL.TXT oligonucleotide" oligonucleotide"

<220> <220> <221> source <221> source <223> /note="Ig Kappa signal peptide" <223> /note="Ig Kappa signal peptide"

<400> 22 <400> 22 atgaggctcc ctgctcagct cctggggctg ctaatgctct gggtcccagg atccagtggg 60 atgaggctcc ctgctcagct cctggggctg ctaatgctct gggtcccagg atccagtggg 60

<210> 23 <210> 23 <211> 54 <211> 54 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="IgE signal peptide" <223> /note="IgE signal peptide"

<400> 23 <400> 23 atggattgga cctggattct gtttctggtg gccgctgcca caagagtgca cagc 54 atggattgga cctggattct gtttctggtg gccgctgcca caagagtgca cagc 54

<210> 24 <210> 24 <211> 63 <211> 63 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="CD8‐alpha signal peptide" <223> /note="CD8-alpha signal peptide"

<400> 24 <400> 24 atggcgctgc ccgtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 atggcgctgc ccgtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63 ccg 63

<210> 25 <210> 25 <211> 63 <211> 63 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence Page 9 Page 9

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic oligonucleotide" oligonucleotide"

<400> 25 <400> 25 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63 ccg 63

<210> 26 <210> 26 <211> 63 <211> 63 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="TVB2(T21A)signal peptide" <223> /note="TVB2(T21A)signal peptide"

<400> 26 <400> 26 atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60 atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60

gct 63 gct 63

<210> 27 <210> 27 <211> 72 <211> 72 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="CD52 signal peptide" <223> /note="CD52 signal peptide"

<400> 27 <400> 27 atgaagcgct tcctcttcct cctactcacc atcagcctcc tggttatggt acagatacaa 60 atgaagcgct tcctcttcct cctactcacc atcagcctcc tggttatggt acagatacaa 60 Page 10 Page 10

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

actggactct ca 72 actggactct ca 72

<210> 28 <210> 28 <211> 84 <211> 84 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> [note="Description of Artificial Sequence: Synthetic oligonucleotide" oligonucleotide"

<220> <220> <221> source <221> source <223> /note="Low‐affinity nerve growth factor receptor <223> /note="Low-affinity nerve growth factor receptor (LNGFR, TNFRSF16) signal peptide" (LNGFR, TNFRSF16) signal peptide"

<400> 28 <400> 28 atgggggcag gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt 60 atgggggcag gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt 60

ctgggggtgt cccttggagg tgcc 84 ctgggggtgt cccttggagg tgcc 84

<210> 29 <210> 29 <211> 141 <211> 141 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="CD8‐alpha hinge region" <223> /note="CD8-alpha hinge region"

<400> 29 <400> 29 aagcccacca ccacccctgc ccctagacct ccaaccccag cccctacaat cgccagccag 60 aagcccacca ccacccctgc ccctagacct ccaaccccag cccctacaat cgccagccag 60

cccctgagcc tgaggcccga agcctgtaga cctgccgctg gcggagccgt gcacaccaga 120 cccctgagcc tgaggcccga agcctgtaga cctgccgctg gcggagccgt gcacaccaga 120

ggcctggatt tcgcctgcga c 141 ggcctggatt tcgcctgcga C 141

<210> 30 <210> 30 <211> 282 <211> 282 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

Page 11 Page 11

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="CD8 alpha 2x" <223> /note="CD8 alpha 2x"

<400> 30 <400> 30 aaacctacta caactcctgc cccccggcct cctacaccag ctcctactat cgcctcccag 60 aaacctacta caactcctgc cccccggcct cctacaccag ctcctactat cgcctcccag 60

ccactcagtc tcagacccga ggcttctagg ccagcggccg gaggcgcggt ccacacccgc 120 ccactcagtc tcagacccga ggcttctagg ccagcggccg gaggcgcggt ccacacccgc 120

gggctggact ttgcatccga taagcccacc accacccctg cccctagacc tccaacccca 180 gggctggact ttgcatccga taagcccacc accacccctg cccctagacc tccaacccca 180

gcccctacaa tcgccagcca gcccctgagc ctgaggcccg aagcctgtag acctgccgct 240 gcccctacaa tcgccagcca gcccctgagc ctgaggcccg aagcctgtag acctgccgct 240

ggcggagccg tgcacaccag aggcctggat ttcgcctgcg ac 282 ggcggagccg tgcacaccag aggcctggat ttcgcctgcg ac 282

<210> 31 <210> 31 <211> 423 <211> 423 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="CD8 alpha 3x" <223> /note="CD8 alpha 3x"

<400> 31 <400> 31 aagcctacca ccacccccgc acctcgtcct ccaacccctg cacctacgat tgccagtcag 60 aagcctacca ccacccccgc acctcgtcct ccaacccctg cacctacgat tgccagtcag 60

cctctttcac tgcggcctga ggccagcaga ccagctgccg gcggtgccgt ccatacaaga 120 cctctttcac tgcggcctga ggccagcaga ccagctgccg gcggtgccgt ccatacaaga 120

ggactggact tcgcgtccga taaacctact accactccag ccccaaggcc cccaacccca 180 ggactggact tcgcgtccga taaacctact accactccag ccccaaggcc cccaacccca 180

gcaccgacta tcgcatcaca gcctttgtca ctgcgtcctg aagccagccg gccagctgca 240 gcaccgacta tcgcatcaca gcctttgtca ctgcgtcctg aagccagccg gccagctgca 240

gggggggccg tccacacaag gggactcgac tttgcgagtg ataagcccac caccacccct 300 gggggggccg tccacacaag gggactcgac tttgcgagtg ataagcccac caccacccct 300

gcccctagac ctccaacccc agcccctaca atcgccagcc agcccctgag cctgaggccc 360 gcccctagac ctccaacccc agcccctaca atcgccagcc agcccctgag cctgaggccc 360

gaagcctgta gacctgccgc tggcggagcc gtgcacacca gaggcctgga tttcgcctgc 420 gaagcctgta gacctgccgc tggcggagcc gtgcacacca gaggcctgga tttcgcctgc 420

gac 423 gac 423

Page 12 Page 12

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<210> 32 <210> 32 <211> 564 <211> 564 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="CD8 alpha 4x" <223> /note="CD8 alpha 4x"

<400> 32 <400> 32 aagcctacca ccacccccgc acctcgtcct ccaacccctg cacctacgat tgccagtcag 60 aagcctacca ccacccccgc acctcgtcct ccaacccctg cacctacgat tgccagtcag 60

gggggggccg tccacacaag gggactcgac tttgcgagtg ataaacctac tacaactcct 300 gggggggccg tccacacaag gggactcgad tttgcgagtg ataaacctac tacaactcct 300

gccccccggc ctcctacacc agctcctact atcgcctccc agccactcag tctcagaccc 360 gccccccggc ctcctacacc agctcctact atcgcctccc agccactcag tctcagaccc 360

gaggcttcta ggccagcggc cggaggcgcg gtccacaccc gcgggctgga ctttgcatcc 420 gaggcttcta ggccagcggc cggaggcgcg gtccacaccc gcgggctgga ctttgcatcc 420

gataagccca ccaccacccc tgcccctaga cctccaaccc cagcccctac aatcgccagc 480 gataagccca ccaccacccc tgcccctaga cctccaaccc cagcccctac aatcgccagc 480

cagcccctga gcctgaggcc cgaagcctgt agacctgccg ctggcggagc cgtgcacacc 540 cagcccctga gcctgaggcc cgaagcctgt agacctgccg ctggcggagc cgtgcacacc 540

agaggcctgg atttcgcctg cgac 564 agaggcctgg atttcgcctg cgac 564

<210> 33 <210> 33 <211> 84 <211> 84 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="CD8‐alpha TM domain" <223> /note="CD8-alpha TM domain"

<400> 33 <400> 33 Page 13 Page 13

50471‐706_601_SL.TXT 50471-706_601_SL.TXT atctacatct gggcccctct ggccggcacc tgtggcgtgc tgctgctgag cctggtcatc 60 atctacatct gggcccctct ggccggcacc tgtggcgtgc tgctgctgag cctggtcatc 60

accctgtact gcaaccaccg gaat 84 accctgtact gcaaccaccg gaat 84

<210> 34 <210> 34 <211> 81 <211> 81 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="CD28 TM domain" <223> /note="CD28 TM domain"

<400> 34 <400> 34 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81 gcctttatta ttttctgggt g 81

<210> 35 <210> 35 <211> 123 <211> 123 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="CD28 signaling domain" <223> /note="CD28 signaling domain"

<400> 35 <400> 35 aggagcaagc ggagcagagg cggccacagc gactacatga acatgacccc ccggaggcct 60 aggagcaage ggagcagagg cggccacaga gactacatga acatgacccc ccggaggcct 60

ggccccaccc ggaagcacta ccagccctac gcccctccca gggacttcgc cgcctaccgg 120 ggccccacco ggaagcacta ccagccctac gcccctccca gggacttcgc cgcctaccgg 120

agc 123 agc 123

<210> 36 <210> 36 <211> 336 <211> 336 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

Page 14 Page 14

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="CD3 zeta signaling domain" <223> /note="CD3 zeta signaling domain"

<400> 36 <400> 36 cgggtgaagt tcagccggag cgccgacgcc cctgcctacc agcagggcca gaaccagctg 60 cgggtgaagt tcagccggag cgccgacgcc cctgcctacc agcagggcca gaaccagctg 60

tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 120 tacaacgago tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 120

cgggaccctg agatgggcgg caagccccgg agaaagaacc ctcaggaggg cctgtataac 180 cgggaccctg agatgggcgg caagccccgg agaaagaacc ctcaggaggg cctgtataac 180

gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240 gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240

cggaggggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggatacc 300 cggaggggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggatacc 300

tacgacgccc tgcacatgca ggccctgccc cccaga 336 tacgacgccc tgcacatgca ggccctgccc cccaga 336

<210> 37 <210> 37 <211> 126 <211> 126 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note=' "Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="4‐1BB signaling domain" <223> /note="4-1BB signaling domain"

<400> 37 <400> 37 aagagaggcc ggaagaaact gctgtacatc ttcaagcagc ccttcatgcg gcccgtgcag 60 aagagaggcc ggaagaaact gctgtacatc ttcaagcago ccttcatgcg gcccgtgcag 60

accacccagg aagaggacgg ctgcagctgc cggttccccg aggaagagga aggcggctgc 120 accacccagg aagaggacgg ctgcagctgc cggttccccg aggaagagga aggcggctgc 120

gaactg 126 gaactg 126

<210> 38 <210> 38 <211> 72 <211> 72 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source Page 15 Page 15

50471‐706_601_SL.TXT 50471-706 601 SL.TXT <223> /note="Description of Artificial Sequence: Synthetic <223> /note= 'Description of Artificial Sequence: Synthetic oligonucleotide" oligonucleotide"

<220> <220> <221> source <221> source <223> /note="DNAX‐activation protein 10 (DAP 10) Signaling <223> /note="DNAX-activation protein 10 (DAP 10) Signaling Domain" Domain"

<400> 38 <400> 38 ctgtgcgcac gcccacgccg cagccccgcc caagaagatg gcaaagtcta catcaacatg 60 ctgtgcgcac gcccacgccg cagccccgcc caagaagatg gcaaagtcta catcaacatg 60

ccaggcaggg gc 72 ccaggcaggg gc 72

<210> 39 <210> 39 <211> 156 <211> 156 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="DNAX‐activation protein 12 (DAP12) Signaling <223> /note="DNAX-activation protein 12 (DAP12) Signaling Domain" Domain"

<400> 39 <400> 39 tacttcctgg gccggctggt ccctcggggg cgaggggctg cggaggcagc gacccggaaa 60 tacttcctgg gccggctggt ccctcggggg cgaggggctg cggaggcage gacccggaaa 60

cagcgtatca ctgagaccga gtcgccttat caggagctcc agggtcagag gtcggatgtc 120 cagcgtatca ctgagaccga gtcgccttat caggagctcc agggtcagag gtcggatgtc 120

tacagcgacc tcaacacaca gaggccgtat tacaaa 156 tacagcgacc tcaacacaca gaggccgtat tacaaa 156

<210> 40 <210> 40 <211> 114 <211> 114 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="IL‐2 core promoter" <223> /note="IL-2 core promoter"

<400> 40 <400> 40 Page 16 Page 16

50471‐706_601_SL.TXT 50471-706_601_SL.TXT acattttgac acccccataa tatttttcca gaattaacag tataaattgc atctcttgtt 60 acattttgac acccccataa tatttttcca gaattaacag tataaattgc atctcttgtt 60

caagagttcc ctatcactct ctttaatcac tactcacagt aacctcaact cctg 114 caagagttcc ctatcactct ctttaatcad tactcacagt aacctcaact cctg 114

<210> 41 <210> 41 <211> 55 <211> 55 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="IL‐2 minimal promoter" <223> /note="IL-2 minimal promoter"

<400> 41 <400> 41 tcaagagttc cctatcactc tctttaatca ctactcacag taacctcaac tcctg 55 tcaagagttc cctatcactc tctttaatca ctactcacag taacctcaac tcctg 55

<210> 42 <210> 42 <211> 380 <211> 380 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="IL‐2 enhancer and promoter variant" <223> /note="IL-2 enhancer and promoter variant"

<400> 42 <400> 42 tgatatcttt tctgagttac ttttgtatcc ccaccccctt aaagaaagga ggaaaaactg 60 tgatatcttt tctgagttac ttttgtatcc ccaccccctt aaagaaagga ggaaaaactg 60

tttcatacag aaggcgttaa ttgcatgaat tagagctatc acctaagtgt gggctaatgt 120 tttcatacag aaggcgttaa ttgcatgaat tagagctatc acctaagtgt gggctaatgt 120

aacaaagagg gatttcacct acatccattc agtcagtctt tgggggttta aagaaattcc 180 aacaaagagg gatttcacct acatccatto agtcagtctt tgggggttta aagaaattcc 180

aaagagtcat cagaagagga aaaatgaagg taatgttttt tcagactggt aaagtctttg 240 aaagagtcat cagaagagga aaaatgaagg taatgttttt tcagactggt aaagtctttg 240

aaaatatgtg taatatgtaa aacattttga cacccccata atatttttcc agaattaaca 300 aaaatatgtg taatatgtaa aacattttga cacccccata atatttttcc agaattaaca 300

gtataaattg catctcttgt tcaagagttc cctatcactc tctttaatca ctactcacag 360 gtataaattg catctcttgt tcaagagttc cctatcactc tctttaatca ctactcacag 360

taacctcaac tcctgccaca 380 taacctcaac tcctgccaca 380

Page 17 Page 17

50471‐706_601_SL.TXT 50471-706_601_SL.TXT -

<210> 43 <210> 43 <211> 373 <211> 373 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<400> 43 <400> 43 ttttctgagt tacttttgta tccccacccc cttaaagaaa ggaggaaaaa ctgtttcata 60 ttttctgagt tacttttgta tccccacccc cttaaagaaa ggaggaaaaa ctgtttcata 60

cagaaggcgt taattgcatg aattagagct atcacctaag tgtgggctaa tgtaacaaag 120 cagaaggcgt taattgcatg aattagagct atcacctaag tgtgggctaa tgtaacaaag 120

agggatttca cctacatcca ttcagtcagt ctttgggggt ttaaagaaat tccaaagagt 180 agggatttca cctacatcca ttcagtcagt ctttgggggt ttaaagaaat tccaaagagt 180

catcagaaga ggaaaaatga aggtaatgtt ttttcagact ggtaaagtct ttgaaaatat 240 catcagaaga ggaaaaatga aggtaatgtt ttttcagact ggtaaagtct ttgaaaatat 240

gtgtaatatg taaaacattt tgacaccccc ataatatttt tccagaatta acagtataaa 300 gtgtaatatg taaaacattt tgacaccccc ataatatttt tccagaatta acagtataaa 300

ttgcatctct tgttcaagag ttccctatca ctctctttaa tcactactca cagtaacctc 360 ttgcatctct tgttcaagag ttccctatca ctctctttaa tcactactca cagtaacctc 360

aactcctgcc aca 373 aactcctgcc aca 373

<210> 44 <210> 44 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note=' 'Description of Artificial Sequence: Synthetic oligonucleotide" oligonucleotide"

<220> <220> <221> source <221> source <223> /note="NF‐kappa‐B binding site" <223> /note="NF-kappa-B binding site"

<400> 44 <400> 44 aagagggatt tcacctacat 20 aagagggatt tcacctacat 20

<210> 45 <210> 45 <211> 11 <211> 11 <212> DNA <212> DNA Page 18 Page 18

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <213> Artificial Sequence <213> Artificial Sequence

<400> 45 <400> 45 aagagggatt t 11 aagagggatt t 11

<210> 46 <210> 46 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<400> 46 <400> 46 agagggattt cacctacatc 20 agagggattt cacctacato 20

<210> 47 <210> 47 <211> 161 <211> 161 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="(NF‐kappa‐B)1‐IL2 promoter variant" <223> /note=" (NF-kappa-B)1-IL2 promoter variant"

<400> 47 <400> 47 aattggtccc atcgaagagg gatttcacct acataattgg tcccgggaca ttttgacacc 60 aattggtccc atcgaagagg gatttcacct acataattgg tcccgggaca ttttgacacc 60

cccataatat ttttccagaa ttaacagtat aaattgcatc tcttgttcaa gagttcccta 120 cccataatat ttttccagaa ttaacagtat aaattgcatc tcttgttcaa gagttcccta 120

Page 19 Page 19

50471‐706_601_SL.TXT 50471-706_601_SL.TXT tcactctctt taatcactac tcacagtaac ctcaactcct g 161 tcactctctt taatcactac tcacagtaac ctcaactcct g 161

<210> 48 <210> 48 <211> 201 <211> 201 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="(NF‐kappa‐B)3‐IL2 promoter variant" <223> /note=" (NF-kappa-B)3-IL2 promoter variant"

<400> 48 <400> 48 aattggtccc atcgaagagg gatttcacct acataagagg gatttcacct acataagagg 60 aattggtccc atcgaagagg gatttcacct acataagagg gatttcacct acataagagg 60

gatttcacct acataattgg tcccgggaca ttttgacacc cccataatat ttttccagaa 120 gatttcacct acataattgg tcccgggaca ttttgacacc cccataatat ttttccagaa 120

ttaacagtat aaattgcatc tcttgttcaa gagttcccta tcactctctt taatcactac 180 ttaacagtat aaattgcatc tcttgttcaa gagttcccta tcactctctt taatcactac 180

tcacagtaac ctcaactcct g 201 tcacagtaac ctcaactcct g 201

<210> 49 <210> 49 <211> 268 <211> 268 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="(NF‐kappa‐B)6‐IL2 promoter variant" <223> /note="(NF-kappa-B)6-IL promoter variant"

<400> 49 <400> 49 aattggtccc atcgaagagg gatttcacct acataagagg gatttcacct acataagagg 60 aattggtccc atcgaagagg gatttcacct acataagagg gatttcacct acataagagg 60

gatttcacct acataattgg taagagggat ttcacctaca taagagggat ttcacctaca 120 gatttcacct acataattgg taagagggat ttcacctaca taagagggat ttcacctaca 120

taagagggat ttcacctaca taattggtcc cgggacattt tgacaccccc ataatatttt 180 taagagggat ttcacctaca taattggtcc cgggacattt tgacaccccc ataatatttt 180

tccagaatta acagtataaa ttgcatctct tgttcaagag ttccctatca ctctctttaa 240 tccagaatta acagtataaa ttgcatctct tgttcaagag ttccctatca ctctctttaa 240

tcactactca cagtaacctc aactcctg 268 tcactactca cagtaacctc aactcctg 268

Page 20 Page 20

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<210> 50 <210> 50 <211> 177 <211> 177 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="1X NFAT Response Elements‐IL2 Promoter variant" <223> /note="1x NFAT Response Elements-IL2 Promoter variant"

<400> 50 <400> 50 aattggtccc atcgaattag gaggaaaaac tgtttcatac agaaggcgtc aattggtccc 60 aattggtccc atcgaattag gaggaaaaac tgtttcatac agaaggcgtc aattggtccc 60

gggacatttt gacaccccca taatattttt ccagaattaa cagtataaat tgcatctctt 120 gggacatttt gacaccccca taatattttt ccagaattaa cagtataaat tgcatctctt 120

gttcaagagt tccctatcac tctctttaat cactactcac agtaacctca actcctg 177 gttcaagagt tccctatcac tctctttaat cactactcac agtaacctca actcctg 177

<210> 51 <210> 51 <211> 36 <211> 36 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Nuclear factor of activated T‐cells (NFAT) <223> /note="Nuclear factor of activated T-cells (NFAT) response element" response element"

<400> 51 <400> 51 aattaggagg aaaaactgtt tcatacagaa ggcgtc 36 aattaggagg aaaaactgtt tcatacagaa ggcgtc 36

<210> 52 <210> 52 <211> 358 <211> 358 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= 'Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

Page 21 Page 21

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <220> <220> <221> source <221> source <223> /note="6X NFAT Response Elements‐IL2 Promoter variant" <223> /note="6x NFAT Response Elements-IL2 Promoter variant"

<400> 52 <400> 52 gaattaggag gaaaaactgt ttcatacaga aggcgtcaat taggaggaaa aactgtttca 60 gaattaggag gaaaaactgt ttcatacaga aggcgtcaat taggaggaaa aactgtttca 60

tacagaaggc gtcaattagg aggaaaaact gtttcataca gaaggcgtca attggtccca 120 tacagaaggc gtcaattagg aggaaaaact gtttcataca gaaggcgtca attggtccca 120

tcgaattagg aggaaaaact gtttcataca gaaggcgtca attaggagga aaaactgttt 180 tcgaattagg aggaaaaact gtttcataca gaaggcgtca attaggagga aaaactgttt 180

catacagaag gcgtcaatta ggaggaaaaa ctgtttcata cagaaggcgt caattggtcc 240 catacagaag gcgtcaatta ggaggaaaaa ctgtttcata cagaaggcgt caattggtco 240

cgggacattt tgacaccccc ataatatttt tccagaatta acagtataaa ttgcatctct 300 cgggacattt tgacaccccc ataatatttt tccagaatta acagtataaa ttgcatctct 300

tgttcaagag ttccctatca ctctccttaa tcactactca cagtaacctc aactcctg 358 tgttcaagag ttccctatca ctctccttaa tcactactca cagtaacctc aactcctg 358

<210> 53 <210> 53 <211> 374 <211> 374 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="6X NFAT Response Elements‐IL2 Promoter variant" <223> /note="6x NFAT Response Elements-IL2 Promoter variant"

<400> 53 <400> 53 tgatatcgaa ttaggaggaa aaactgtttc atacagaagg cgtcaattag gaggaaaaac 60 tgatatcgaa ttaggaggaa aaactgtttc atacagaagg cgtcaattag gaggaaaaao 60

tgtttcatac agaaggcgtc aattaggagg aaaaactgtt tcatacagaa ggcgtcaatt 120 tgtttcatad agaaggcgtc aattaggagg aaaaactgtt tcatacagaa ggcgtcaatt 120

ggtcccatcg aattaggagg aaaaactgtt tcatacagaa ggcgtcaatt aggaggaaaa 180 ggtcccatcg aattaggagg aaaaactgtt tcatacagaa ggcgtcaatt aggaggaaaa 180

actgtttcat acagaaggcg tcaattagga ggaaaaactg tttcatacag aaggcgtcaa 240 actgtttcat acagaaggcg tcaattagga ggaaaaactg tttcatacag aaggcgtcaa 240

ttggtcccgg gacattttga cacccccata atatttttcc agaattaaca gtataaattg 300 ttggtcccgg gacattttga cacccccata atatttttcc agaattaaca gtataaattg 300

catctcttgt tcaagagttc cctatcactc tctttaatca ctactcacag taacctcaac 360 catctcttgt tcaagagttc cctatcactc tctttaatca ctactcacag taacctcaac 360

tcctgaattc catg 374 tcctgaattc catg 374

<210> 54 <210> 54 <211> 358 <211> 358 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence Page 22 Page 22

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<400> 54 <400> 54 gaattaggag gaaaaactgt ttcatacaga aggcgtcaat taggaggaaa aactgtttca 60 gaattaggag gaaaaactgt ttcatacaga aggcgtcaat taggaggaaa aactgtttca 60

catacagaag gcgtcaatta ggaggaaaaa ctgtttcata cagaaggcgt caattggtcc 240 catacagaag gcgtcaatta ggaggaaaaa ctgtttcata cagaaggcgt caattggtcc 240

tgttcaagag ttccctatca ctctctttaa tcactactca cagtaacctc aactcctg 358 tgttcaagag ttccctatca ctctctttaa tcactactca cagtaacctc aactcctg 358

<210> 55 <210> 55 <211> 365 <211> 365 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<400> 55 <400> 55 tgatatcgaa ttaggaggaa aaactgtttc atacagaagg cgtcaattag gaggaaaaac 60 tgatatcgaa ttaggaggaa aaactgtttd atacagaagg cgtcaattag gaggaaaaac 60

tgtttcatac agaaggcgtc aattaggagg aaaaactgtt tcatacagaa ggcgtcaatt 120 tgtttcatac agaaggcgtc aattaggagg aaaaactgtt tcatacagaa ggcgtcaatt 120

catctcttgt tcaagagttc cctatcactc tctttaatca ctactcacag taacctcaac 360 catctcttgt tcaagagttc cctatcacto tctttaatca ctactcacag taacctcaac 360

tcctg 365 tcctg 365 Page 23 Page 23

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<210> 56 <210> 56 <211> 256 <211> 256 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="3X NFAT Response Elements‐IL2 Promoter variant" <223> /note="3X NFAT Response Elements-IL2 - Promoter variant"

<400> 56 <400> 56 tgatatcaat tggtcccatc gaattaggag gaaaaactgt ttcatacaga aggcgtcaat 60 tgatatcaat tggtcccatc gaattaggag gaaaaactgt ttcatacaga aggcgtcaat 60

taggaggaaa aactgtttca tacagaaggc gtcaattagg aggaaaaact gtttcataca 120 taggaggaaa aactgtttca tacagaaggc gtcaattagg aggaaaaact gtttcataca 120

gaaggcgtca attggtcccg ggacattttg acacccccat aatatttttc cagaattaac 180 gaaggcgtca attggtcccg ggacattttg acacccccat aatatttttc cagaattaac 180

agtataaatt gcatctcttg ttcaagagtt ccctatcact ctctttaatc actactcaca 240 agtataaatt gcatctcttg ttcaagagtt ccctatcact ctctttaatc actactcaca 240

gtaacctcaa ctcctg 256 gtaacctcaa ctcctg 256

<210> 57 <210> 57 <211> 249 <211> 249 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<400> 57 <400> 57 aattggtccc atcgaattag gaggaaaaac tgtttcatac agaaggcgtc aattaggagg 60 aattggtccc atcgaattag gaggaaaaac tgtttcatac agaaggcgtc aattaggagg 60

aaaaactgtt tcatacagaa ggcgtcaatt aggaggaaaa actgtttcat acagaaggcg 120 aaaaactgtt tcatacagaa ggcgtcaatt aggaggaaaa actgtttcat acagaaggcg 120

tcaattggtc ccgggacatt ttgacacccc cataatattt ttccagaatt aacagtataa 180 tcaattggtc ccgggacatt ttgacacccc cataatattt ttccagaatt aacagtataa 180

attgcatctc ttgttcaaga gttccctatc actctcttta atcactactc acagtaacct 240 attgcatctc ttgttcaaga gttccctatc actctcttta atcactacto acagtaacct 240

caactcctg 249 caactcctg 249 Page 24 Page 24

50471‐706_601_SL.TXT 50471-706_601_SL.TXT -

<210> 58 <210> 58 <211> 244 <211> 244 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<220> <220> <221> source <221> source <223> /note="human EEF1A1 promoter variant" <223> /note="human EEF1A1 promoter variant"

<400> 58 <400> 58 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagago gcacatcgcc cacagtcccc 60

gagaagttgg ggggaggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt 120 gagaagttgg ggggaggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt 120

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180

gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240

acag 244 acag 244

<210> 59 <210> 59 <211> 236 <211> 236 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 59 <400> 59 gcgtgaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag 60 gcgtgaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag 60

ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg 120 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg 120

gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata 180 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata 180

agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacaca 236 agtgcagtag tcgccgtgaa cgttcttttt cgcaaccgggt ttgccgccag aacaca 236

<210> 60 <210> 60 <211> 1266 <211> 1266 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<220> <220> <221> source <221> source <223> /note="human EEF1A1 promoter and enhancer" <223> /note="human EEF1A1 promoter and enhancer"

<400> 60 <400> 60 Page 25 Page 25

50471‐706_601_SL.TXT gagctttgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct 60 60

tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat 120 120

cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa 180 180

ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg 240

gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt 300

ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 360

gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc 420 420

ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt 480

cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt 540 540

ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat 600

gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc 660

acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca 720

catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc 780 780

aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg 840 840

cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc 900

ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac 960 960

ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt 1020 1020

accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag 1080

gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag 1140 1140

ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat 1200

cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt 1260

1266 cgtgag 1266

<210> <210> 61 <211> 571 <212> <212> DNA <213> Homo sapiens

<220> <220> <221> <221> source Page 26 page 26

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <223> /note="Human UBC promoter" <223> /note="Humar UBC promoter"

<400> 61 <400> 61 ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg 60 ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg 60

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag 120 ccacgtcaga cgaagggcgc agcgagcgtc ctgatcctto cgcccggacg ctcaggacag 120

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag 180 cggcccgctg ctcataagad tcggccttag aaccccagta tcagcagaag gacattttag 180

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg 240 gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg 240

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat 300 aaaagtagtc ccttctcggc gattctgcgg agggatctco gtggggcggt gaacgccgat 300

gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt 360 gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt 360

cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct 420 cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct 420

gggtacgtgc gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa 480 gggtacgtgc gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa 480

atgtaatcat ttgggtcaat atgtaatttt cagtgttaga ctagtaaatt gtccgctaaa 540 atgtaatcat ttgggtcaat atgtaatttt cagtgttaga ctagtaaatt gtccgctaaa 540

ttctggccgt ttttggcttt tttgttagac g 571 ttctggccgt ttttggcttt tttgttagad g 571

<210> 62 <210> 62 <211> 148 <211> 148 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note=" "Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="6 site GAL4‐inducible proximal factor binding <223> /note="6 site GAL4-inducible proximal factor binding element (PFB)" element (PFB)"

<400> 62 <400> 62 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60

gactattccg gggtttaccg gcgcactctc gcccgaactt caccggcggt ctttcgtccg 120 gactattccg gggtttaccg gcgcactctc gcccgaactt caccggcggt ctttcgtccg 120

tgctttatcg gggcggatca ctccgaac 148 tgctttatcg gggcggatca ctccgaac 148

<210> 63 <210> 63 <211> 81 <211> 81 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

Page 27 Page 27

50471‐706_601_SL.TXT 50471-706_601_SL.TXT - <220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" oligonucleotide"

<220> <220> <221> source <221> source <223> /note="Synthetic minimal promoter 1 [Inducible Promoter]" <223> /note="Synthetic minimal promoter 1 [Inducible Promoter]"

<400> 63 <400> 63 aggtctatat aagcagagct cgtttagtga accctcattc tggagacgga tcccgagccg 60 aggtctatat aagcagagct cgtttagtga accctcattc tggagacgga tcccgagccg 60

agtgttttga cctccataga a 81 agtgttttga cctccataga a 81

<210> 64 <210> 64 <211> 31 <211> 31 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Synthetic 5' UTR based on RPL6" <223> /note="Synthetic 5' UTR based on RPL6"

<400> 64 <400> 64 cagccgctaa atccaaggta aggtcagaag a 31 cagccgctaa atccaaggta aggtcagaag a 31

<210> 65 <210> 65 <211> 132 <211> 132 <212> DNA <212> DNA <213> Simian virus 40 <213> Simian virus 40

<220> <220> <221> source <221> source <223> /note="SV40e polyA" <223> /note="SV40e polyA"

<400> 65 <400> 65 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

tatcatgtct gg 132 tatcatgtct gg 132

<210> 66 <210> 66 <211> 275 <211> 275 Page 28 Page 28

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Bidirectional aCA polyA [bidirectional polyA]" <223> /note="Bidirectional aCA polyA [bidirectional polyA]"

<400> 66 <400> 66 atcgattaat ctagcggccc tagacgagca gacatgataa gatacattga tgagtttgga 60 atcgattaat ctagcggccc tagacgagca gacatgataa gatacattga tgagtttgga 60

caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt 120 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt 120

gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 180 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 180

tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac 240 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac 240

aaatgtggta aaatccgata agcgtaccta gaggc 275 aaatgtggta aaatccgata agcgtaccta gaggc 275

<210> 67 <210> 67 <211> 57 <211> 57 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="PA2 polyA" <223> /note="PA2 polyA"

<400> 67 <400> 67 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgag 57 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgag 57

<210> 68 <210> 68 <211> 258 <211> 258 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note=' 'Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

Page 29 Page 29

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <220> <220> <221> source <221> source <223> /note="VP16 activation domain" <223> /note="VP16 activation domain'

<400> 68 <400> 68 ggccccaaga agaaaaggaa ggtggccccc cccaccgacg tgagcctggg cgacgagctg ggccccaaga agaaaaggaa ggtggccccc cccaccgacg tgagcctggg cgacgagctg 60 60

cacctggacg gcgaggacgt ggccatggcc cacgccgacg ccctggacga cttcgacctg cacctggacg gcgaggacgt ggccatggcc cacgccgacg ccctggacga cttcgacctg 120 120

gacatgctgg gcgacggcga cagccccggc cccggcttca ccccccacga cagcgccccc gacatgctgg gcgacggcga cagccccggc cccggcttca ccccccacga cagcgccccc 180 180

tacggcgccc tggacatggc cgacttcgag ttcgagcaga tgttcaccga cgccctgggc tacggcgccc tggacatggc cgacttcgag ttcgagcaga tgttcaccga cgccctgggc 240 240

atcgacgagt acggcggc 258 atcgacgagt acggcggc 258

<210> 69 <210> 69 <211> 705 <211> 705 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note= 'Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide'

<220> <220> <221> source <221> source <223> /note="Retinoid x receptor (RxR)" <223> /note="Retinoid X receptor (RxR) "

<400> 69 <400> 69 gagatgcccg tggacaggat tctggaggcc gaactcgccg tggagcagaa aagcgaccag gagatgcccg tggacaggat tctggaggcc gaactcgccg tggagcagaa aagcgaccag 60 60

ggcgtggagg gccccggcgg aaccggcggc agcggcagca gccccaacga ccccgtgacc ggcgtggagg gccccggcgg aaccggcggc agcggcagca gccccaacga ccccgtgacc 120 120

aacatctgcc aggccgccga caagcagctg ttcaccctgg tggagtgggc caagaggatt aacatctgcc aggccgccga caagcagctg ttcaccctgg tggagtgggc caagaggatt 180 180

ccccacttca gcagcctgcc cctggacgad caggtgatcc tgctgagggo cggatggaad ccccacttca gcagcctgcc cctggacgac caggtgatcc tgctgagggc cggatggaac 240 240

gagctgctga tcgccagctt cagccacagg agcatcgacg tgagggacgg catcctgctg gagctgctga tcgccagctt cagccacagg agcatcgacg tgagggacgg catcctgctg 300 300

gccaccggcc tgcacgtcca taggaacago gcccacagcg ccggagtggg cgccatctto gccaccggcc tgcacgtcca taggaacagc gcccacagcg ccggagtggg cgccatcttc 360 360

gacagggtgo tgaccgagct ggtgagcaag atgagggaca tgaggatgga caagaccgag gacagggtgc tgaccgagct ggtgagcaag atgagggaca tgaggatgga caagaccgag 420 420

ctgggctgcc tgagggccat catcctgttc aaccccgagg tgaggggcct gaaaagcgcc ctgggctgcc tgagggccat catcctgttc aaccccgagg tgaggggcct gaaaagcgcc 480 480

caggaggtgg agctgctgag ggagaaggtg tacgccgccc tggaggagta caccaggaco caggaggtgg agctgctgag ggagaaggtg tacgccgccc tggaggagta caccaggacc 540 540

acccaccccg acgagcccgg cagattcgcc aagctgctgc tgaggctgco cagcctgagg acccaccccg acgagcccgg cagattcgcc aagctgctgc tgaggctgcc cagcctgagg 600 600

agcatcggcc tgaagtgcct ggagcacctg ttcttcttca ggctgatcgg cgacgtgccc agcatcggcc tgaagtgcct ggagcacctg ttcttcttca ggctgatcgg cgacgtgccc 660 660

Page 30 Page 30

50471‐706_601_SL.TXT

atcgacacct tcctgatgga gatgctggag agccccagcg acagc 705 SOL

<210> 70 OL <0IZ> <211> 969 696 <IIZ> <212> DNA ANC <ZIZ> <213> Artificial Sequence <ETZ>

<220> <022> <221> source <IZZ> <223> /note="Description of Artificial Sequence: Synthetic

JO <EZZ> polynucleotide"

<220> <022> <221> source <IZZ> <223> /note="VP16‐linker‐RxR"

/ <EZZ> <400> 70 OL <00 ggccccaaga agaaaaggaa ggtggccccc cccaccgacg tgagcctggg cgacgagctg 60 09 eece cacctggacg gcgaggacgt ggccatggcc cacgccgacg ccctggacga cttcgacctg 120 OZI

gacatgctgg gcgacggcga cagccccggc cccggcttca ccccccacga cagcgccccc 180 08T

tacggcgccc tggacatggc cgacttcgag ttcgagcaga tgttcaccga cgccctgggc 240

atcgacgagt acggcggcga attcgagatg cccgtggaca ggattctgga ggccgaactc 300 00E 2000 gccgtggagc agaaaagcga ccagggcgtg gagggccccg gcggaaccgg cggcagcggc 360 09E

agcagcccca acgaccccgt gaccaacatc tgccaggccg ccgacaagca gctgttcacc 420

7 e ctggtggagt gggccaagag gattccccac ttcagcagcc tgcccctgga cgaccaggtg 480 08/

atcctgctga gggccggatg gaacgagctg ctgatcgcca gcttcagcca caggagcatc 540

gacgtgaggg acggcatcct gctggccacc ggcctgcacg tccataggaa cagcgcccac 600 009

agcgccggag tgggcgccat cttcgacagg gtgctgaccg agctggtgag caagatgagg 660 099

gacatgagga tggacaagac cgagctgggc tgcctgaggg ccatcatcct gttcaacccc 720 02L

gaggtgaggg gcctgaaaag cgcccaggag gtggagctgc tgagggagaa ggtgtacgcc 780 08L

e gccctggagg agtacaccag gaccacccac cccgacgagc ccggcagatt cgccaagctg 840

ctgctgaggc tgcccagcct gaggagcatc ggcctgaagt gcctggagca cctgttcttc 900 006

ttcaggctga tcggcgacgt gcccatcgac accttcctga tggagatgct ggagagcccc 960 096

agcgacagc 969 Page 31 696

TE aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<210> 71 <210> 71 <211> 450 <211> 450 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note=" 'Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="GAL4 DNA Binding Domain" <223> /note="GAL4 DNA Binding Domain"

<400> 71 <400> 71 atgaagctgc tgagcagcat cgagcaggct tgcgacatct gcaggctgaa gaagctgaag 60 atgaagctgc tgagcagcat cgagcaggct tgcgacatct gcaggctgaa gaagctgaag 60

tgcagcaagg agaagcccaa gtgcgccaag tgcctgaaga acaactggga gtgcagatac 120 tgcagcaagg agaagcccaa gtgcgccaag tgcctgaaga acaactggga gtgcagatac 120

agccccaaga ccaagaggag ccccctgacc agggcccacc tgaccgaggt ggagagcagg 180 agccccaaga ccaagaggag ccccctgacc agggcccacc tgaccgaggt ggagagcagg 180

ctggagaggc tggagcagct gttcctgctg atcttcccca gggaggacct ggacatgatc 240 ctggagaggc tggagcagct gttcctgctg atcttcccca gggaggacct ggacatgatc 240

ctgaagatgg acagcctgca agacatcaag gccctgctga ccggcctgtt cgtgcaggac 300 ctgaagatgg acagcctgca agacatcaag gccctgctga ccggcctgtt cgtgcaggad 300

aacgtgaaca aggacgccgt gaccgacagg ctggccagcg tggagaccga catgcccctg 360 aacgtgaaca aggacgccgt gaccgacagg ctggccagcg tggagaccga catgcccctg 360

accctgaggc agcacaggat cagcgccacc agcagcagcg aggagagcag caacaagggc 420 accctgaggc agcacaggat cagcgccacc agcagcagcg aggagagcag caacaagggc 420

cagaggcagc tgaccgtgag ccccgagttt 450 cagaggcage tgaccgtgag ccccgagttt 450

<210> 72 <210> 72 <211> 1008 <211> 1008 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Ecdysone Receptor Ligand Binding Domain <223> /note="Ecdysone Receptor Ligand Binding Domain ‐ VY variant (EcR)" - VY variant (EcR)

<400> 72 <400> 72 atcaggcccg agtgcgtggt gcccgagacc cagtgcgcca tgaaaaggaa ggagaagaag 60 atcaggcccg agtgcgtggt gcccgagacc cagtgcgcca tgaaaaggaa ggagaagaag 60

Page 32 Page 32

50471‐706_601_SL.TXT 50471-706_601_SL.TXT gcccagaagg agaaggacaa gctgcccgtg agcaccacca ccgtcgatga ccacatgccc 120 gcccagaagg agaaggacaa gctgcccgtg agcaccacca ccgtcgatga ccacatgccc 120

cccatcatgc agtgcgagcc cccccccccc gaggccgcca ggattcacga ggtcgtgccc 180 cccatcatgc agtgcgagcc CCCCCCCCCCC gaggccgcca ggattcacga ggtcgtgccc 180

aggttcctga gcgacaagct gctggtgacc aacaggcaga agaacatccc ccagctgacc 240 aggttcctga gcgacaagct gctggtgacc aacaggcaga agaacatccc ccagctgacc 240

gccaaccagc agttcctgat cgccaggctg atctggtatc aggacggcta cgagcagccc 300 gccaaccagc agttcctgat cgccaggctg atctggtatc aggacggcta cgagcagccc 300

agcgacgagg acctgaaaag gatcacccag acctggcagc aggccgacga cgagaacgag 360 agcgacgagg acctgaaaag gatcacccag acctggcagc aggccgacga cgagaacgag 360

gagagcgaca cccccttcag gcagatcacc gagatgacca tcctgaccgt gcagctgatc 420 gagagcgaca cccccttcag gcagatcacc gagatgacca tcctgaccgt gcagctgatc 420

gtggagttcg ccaagggcct gcccggattc gccaagatca gccagcccga ccagatcacc 480 gtggagttcg ccaagggcct gcccggattc gccaagatca gccagcccga ccagatcacc 480

ctgctgaagg cttgcagcag cgaggtgatg atgctgaggg tggccaggag gtacgacgcc 540 ctgctgaagg cttgcagcag cgaggtgatg atgctgaggg tggccaggag gtacgacgcc 540

gccagcgaca gcatcctgtt cgccaacaac caggcttaca ccagggacaa ctacaggaag 600 gccagcgaca gcatcctgtt cgccaacaac caggettaca ccagggacaa ctacaggaag 600

gctggcatgg ccgaggtgat cgaggacctc ctgcacttct gcagatgtat gtacagcatg 660 gctggcatgg ccgaggtgat cgaggacctc ctgcacttct gcagatgtat gtacagcatg 660

gccctggaca acatccacta cgccctgctg accgccgtgg tgatcttcag cgacaggccc 720 gccctggaca acatccacta cgccctgctg accgccgtgg tgatcttcag cgacaggccc 720

ggcctggagc agccccagct ggtggaggag atccagaggt actacctgaa caccctgagg 780 ggcctggago agccccagct ggtggaggag atccagaggt actacctgaa caccctgagg 780

atctacatcc tgaaccagct gagcggcagc gccaggagca gcgtgatcta cggcaagatc 840 atctacatcc tgaaccagct gagcggcagc gccaggagca gcgtgatcta cggcaagato 840

ctgagcatcc tgagcgagct gaggaccctg ggaatgcaga acagcaatat gtgtatcagc 900 ctgagcatcc tgagcgagct gaggaccctg ggaatgcaga acagcaatat gtgtatcagc 900

ctgaagctga agaacaggaa gctgcccccc ttcctggagg agatttggga cgtggccgac 960 ctgaagctga agaacaggaa gctgcccccc ttcctggagg agatttggga cgtggccgac 960

atgagccaca cccagccccc ccccatcctg gagagcccca ccaacctg 1008 atgagccaca cccagccccc ccccatcctg gagagcccca ccaacctg 1008

<210> 73 <210> 73 <211> 1008 <211> 1008 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Ecdysone Receptor Ligand Binding Domain <223> /note="Ecdysone Receptor Ligand Binding Domain ‐ VY variant (EcR)" - VY variant (EcR)" "

<400> 73 <400> 73 cggcctgagt gcgtagtacc cgagactcag tgcgccatga agcggaaaga gaagaaagca 60 cggcctgagt gcgtagtacc cgagactcag tgcgccatga agcggaaaga gaagaaagca 60

cagaaggaga aggacaaact gcctgtcagc acgacgacgg tggacgacca catgccgccc 120 cagaaggaga aggacaaact gcctgtcagc acgacgacgg tggacgacca catgccgccc 120

Page 33 Page 33

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

attatgcagt gtgaacctcc acctcctgaa gcagcaagga ttcacgaagt ggtcccaagg 180 attatgcagt gtgaacctcc acctcctgaa gcagcaagga ttcacgaagt ggtcccaagg 180

tttctctccg acaagctgtt ggtgacaaac cggcagaaaa acatccccca gttgacagcc 240 tttctctccg acaagctgtt ggtgacaaac cggcagaaaa acatccccca gttgacagco 240

aaccagcagt tccttatcgc caggctcatc tggtaccagg acgggtacga gcagccttct 300 aaccagcagt tccttatcgc caggetcato tggtaccagg acgggtacga gcagccttct 300

gatgaagatt tgaagaggat tacgcagacg tggcagcaag cggacgatga aaacgaagag 360 gatgaagatt tgaagaggat tacgcagacg tggcagcaag cggacgatga aaacgaagag 360

tcggacactc ccttccgcca gatcacagag atgactatcc tcacggtcca acttatcgtg 420 tcggacacto ccttccgcca gatcacagag atgactatcc tcacggtcca acttatcgtg 420

gagttcgcga agggattgcc agggttcgcc aagatctcgc agcctgatca aattacgctg 480 gagttcgcga agggattgcc agggttcgcc aagatctcgc agcctgatca aattacgctg 480

cttaaggctt gctcaagtga ggtaatgatg ctccgagtcg cgcgacgata cgatgcggcc 540 cttaaggctt gctcaagtga ggtaatgatg ctccgagtcg cgcgacgata cgatgcggcc 540

tcagacagta ttctgttcgc gaacaaccaa gcgtacactc gcgacaacta ccgcaaggct 600 tcagacagta ttctgttcgc gaacaaccaa gcgtacactc gcgacaacta ccgcaaggct 600

ggcatggccg aggtcatcga ggatctactg cacttctgcc ggtgcatgta ctctatggcg 660 ggcatggccg aggtcatcga ggatctactg cacttctgcc ggtgcatgta ctctatggcg 660

ttggacaaca tccattacgc gctgctcacg gctgtcgtca tcttttctga ccggccaggg 720 ttggacaaca tccattacgc gctgctcacg gctgtcgtca tcttttctga ccggccaggg 720

ttggagcagc cgcaactggt ggaagagatc cagcggtact acctgaatac gctccgcatc 780 ttggagcago cgcaactggt ggaagagatc cagcggtact acctgaatad gctccgcatc 780

tatatcctga accagctgag cgggtcggcg cgttcgtccg tcatatacgg caagatcctc 840 tatatcctga accagctgag cgggtcggcg cgttcgtccg tcatatacgg caagatcctc 840

tcaatcctct ctgagctacg cacgctcggc atgcaaaact ccaacatgtg catctccctc 900 tcaatcctct ctgagctacg cacgctcggc atgcaaaact ccaacatgtg catctccctc 900

aagctcaaga acagaaagct gccgcctttc ctcgaggaga tctgggatgt ggcggacatg 960 aagctcaaga acagaaagct gccgcctttc ctcgaggaga tctgggatgt ggcggacatg 960

tcgcacaccc aaccgccgcc tatcctcgag tcccccacga atctctag 1008 tcgcacacco aaccgccgcc tatcctcgag tcccccacga atctctag 1008

<210> 74 <210> 74 <211> 1467 <211> 1467 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="GAL4‐Linker‐EcR" <223> /note="GAL4-Linker-EcR"

<400> 74 <400> 74 atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag 60 atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag 60

tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac 120 tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac 120

tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg 180 tctcccaaaa ccaaaaggtc tccgctgact agggcacato tgacagaagt ggaatcaagg 180

Page 34 Page 34

50471‐706_601_SL.TXT

ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt 240

ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat 300

aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta 360

acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt 420

caaagacagt tgactgtatc gccggaattc ccggggatcc ggcctgagtg cgtagtaccc 480

gagactcagt gcgccatgaa gcggaaagag aagaaagcac agaaggagaa ggacaaactg 540

cctgtcagca cgacgacggt ggacgaccac atgccgccca ttatgcagtg tgaacctcca 600

cctcctgaag cagcaaggat tcacgaagtg gtcccaaggt ttctctccga caagctgttg 660

gtgacaaacc ggcagaaaaa catcccccag ttgacagcca accagcagtt ccttatcgcc 720

aggctcatct ggtaccagga cgggtacgag cagccttctg atgaagattt gaagaggatt 780

acgcagacgt ggcagcaagc ggacgatgaa aacgaagagt cggacactcc cttccgccag 840

atcacagaga tgactatcct cacggtccaa cttatcgtgg agttcgcgaa gggattgcca 900

gggttcgcca agatctcgca gcctgatcaa attacgctgc ttaaggcttg ctcaagtgag 960

gtaatgatgc tccgagtcgc gcgacgatac gatgcggcct cagacagtat tctgttcgcg 1020

aacaaccaag cgtacactcg cgacaactac cgcaaggctg gcatggccga ggtcatcgag 1080

gatctactgc acttctgccg gtgcatgtac tctatggcgt tggacaacat ccattacgcg 1140

ctgctcacgg ctgtcgtcat cttttctgac cggccagggt tggagcagcc gcaactggtg 1200

gaagagatcc agcggtacta cctgaatacg ctccgcatct atatcctgaa ccagctgagc 1260

gggtcggcgc gttcgtccgt catatacggc aagatcctct caatcctctc tgagctacgc 1320 ao

acgctcggca tgcaaaactc caacatgtgc atctccctca agctcaagaa cagaaagctg 1380 00

ccgcctttcc tcgaggagat ctgggatgtg gcggacatgt cgcacaccca accgccgcct 1440

atcctcgagt cccccacgaa tctctag 1467

<210> 75 <211> 1464 <212> DNA <213> Artificial Sequence

<220> Page 35

50471‐706_601_SL.TXT <221> source acunos <IZZ> <223> /note="Description of Artificial Sequence: Synthetic

JO <EZZ> polynucleotide"

<220> <022> <221> source <IZZ> <223> /note="GAL4‐Linker‐EcR"

0/ <EZZ> <400> 75 SL <00t>> atgaagctgc tgagcagcat cgagcaggct tgcgacatct gcaggctgaa gaagctgaag 60 09

tgcagcaagg agaagcccaa gtgcgccaag tgcctgaaga acaactggga gtgcagatac 120

agccccaaga ccaagaggag ccccctgacc agggcccacc tgaccgaggt ggagagcagg 180 08T

e e ctggagaggc tggagcagct gttcctgctg atcttcccca gggaggacct ggacatgatc 240

ctgaagatgg acagcctgca agacatcaag gccctgctga ccggcctgtt cgtgcaggac 300 7787508800 00E

aacgtgaaca aggacgccgt gaccgacagg ctggccagcg tggagaccga catgcccctg 360 09E

accctgaggc agcacaggat cagcgccacc agcagcagcg aggagagcag caacaagggc 420

credit 7 cagaggcagc tgaccgtgag ccccgagttt cccgggcggc ctgagtgcgt agtacccgag 480 08/

actcagtgcg ccatgaagcg gaaagagaag aaagcacaga aggagaagga caaactgcct 540 essee SeedeSeee8 gtcagcacga cgacggtgga cgaccacatg ccgcccatta tgcagtgtga acctccacct 600 009

cctgaagcag caaggattca cgaagtggtc ccaaggtttc tctccgacaa gctgttggtg 660 099

acaaaccggc agaaaaacat cccccagttg acagccaacc agcagttcct tatcgccagg 720 OZL

ctcatctggt accaggacgg gtacgagcag ccttctgatg aagatttgaa gaggattacg 780 08L

cagacgtggc agcaagcgga cgatgaaaac gaagagtcgg acactccctt ccgccagatc 840

acagagatga ctatcctcac ggtccaactt atcgtggagt tcgcgaaggg attgccaggg 900 006

ttcgccaaga tctcgcagcc tgatcaaatt acgctgctta aggcttgctc aagtgaggta 960 096

atgatgctcc gagtcgcgcg acgatacgat gcggcctcag acagtattct gttcgcgaac 1020 0201

aaccaagcgt acactcgcga caactaccgc aaggctggca tggccgaggt catcgaggat 1080 080T

ctactgcact tctgccggtg catgtactct atggcgttgg acaacatcca ttacgcgctg 1140

ctcacggctg tcgtcatctt ttctgaccgg ccagggttgg agcagccgca actggtggaa 1200

gagatccagc ggtactacct gaatacgctc cgcatctata tcctgaacca gctgagcggg 1260 092T

tcggcgcgtt cgtccgtcat atacggcaag atcctctcaa tcctctctga gctacgcacg 1320 OZET

thePage 36 9E aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

ctcggcatgo aaaactccaa catgtgcato tccctcaagc tcaagaacag aaagctgccg ctcggcatgc aaaactccaa catgtgcatc tccctcaagc tcaagaacag aaagctgccg 1380 1380

cctttcctcg aggagatctg ggatgtggcg gacatgtcgc acacccaaco gccgcctatc cctttcctcg aggagatctg ggatgtggcg gacatgtcgc acacccaacc gccgcctatc 1440 1440

ctcgagtccc ccacgaatct ctag 1464 ctcgagtccc ccacgaatct ctag 1464

<210> 76 <210> 76 <211> 1005 <211> 1005 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note=" "Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="Truncated EGFR (huEGFRt) (Her1t)" <223> /note="Truncated EGFR (huEGFRt) (Her1t) "

<400> 76 <400> 76 cgcaaaattgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 60

acgaatatta aacacttcaa aaactgcaco tccatcagtg gcgatctcca catcctgccg acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 120

gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180 180

attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240 240

aggacggacc tccatgcctt tgagaaccta gaaatcatad gcggcaggad caagcaacat aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 300

ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360 360

aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420 420

acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480 480

agaggtgaaa acagctgcaa ggccacaggo caggtctgcc atgccttgtg ctcccccgag agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 540

ggctgctggg gcccggagco cagggactgo gtctcttgcc ggaatgtcag ccgaggcagg ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600 600

gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 660 660

gagtgcatad agtgccacco agagtgcctg cctcaggcca tgaacatcad ctgcacagga gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720 720

cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag 780 cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag 780

acctgcccgg caggagtcat gggagaaaao aacaccctgg tctggaagta cgcagacgcc acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 840 840

ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt 900 900

Page 37 Page 37

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

gaaggctgtc caacgaatgg gcctaagatc ccgtccatcg ccactgggat ggtgggggcc 960 gaaggctgtc caacgaatgg gcctaagatc ccgtccatcg ccactgggat ggtgggggcc 960

ctcctcttgc tgctggtggt ggccctgggg atcggcctct tcatg 1005 ctcctcttgc tgctggtggt ggccctggggg atcggcctct tcatg 1005

<210> 77 <210> 77 <211> 732 <211> 732 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 1 (Her1 truncated design 1) <223> /note="EGFR truncated design 1 (Her1 truncated design 1) (HER1t1)" (HER1t1)"

<400> 77 <400> 77 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 cgcaaaatt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120

gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180 gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180

attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240 attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240

aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300

ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360 ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360

aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420 aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420

acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480 acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480

agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540

ggctgctggg gcccggagcc cagggactgc gtctctggtg gcggtggctc gggcggtggt 600 ggctgctggg gcccggagcc cagggactgo gtctctggtg gcggtggctc gggcggtggt 600

gggtcgggtg gcggcggatc tggtggcggt ggctcgtttt gggtgctggt ggtggttggt 660 gggtcgggtg gcggcggatc tggtggcggt ggctcgtttt gggtgctggt ggtggttggt 660

ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 720 ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 720

agtaagagga gc 732 agtaagagga gc 732

<210> 78 <210> 78 <211> 771 <211> 771 <212> DNA <212> DNA Page 38 Page 38

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 2 (Her1 truncated design 2) <223> /note="EGFR truncated design 2 (Her1 truncated design 2) (HER1t2)" (HER1t2)'

<400> 78 <400> 78 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 cgcaaaattgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600 ggctgctggg gcccggagco cagggactgo gtctcttgcc ggaatgtcag ccgaggcagg 600

gaatgcgtgg acaagggtgg cggtggctcg ggcggtggtg ggtcgggtgg cggcggatct 660 gaatgcgtgg acaagggtgg cggtggctcg ggcggtggtg ggtcgggtgg cggcggatct 660

ggtggcggtg gctcgttttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 720 ggtggcggtg gctcgttttg ggtgctggtg gtggttggtg gagtcctggc ttgctatago 720

ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag c 771 ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag C 771

<210> 79 <210> 79 <211> 828 <211> 828 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source Page 39 Page 39

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <223> /note="EGFR truncated design 3 (Her1 truncated design 3) <223> /note="EGFR truncated design 3 (Her1 truncated design 3) (HER1t3)" (HER1t3)'

<400> 79 <400> 79 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 cgcaaaattgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 acgaatatta aacacttcaa aaactgcaco tccatcagtg gcgatctcca catcctgccg 120

ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600 ggctgctggg gcccggagcc cagggactgo gtctcttgcc ggaatgtcag ccgaggcagg 600

gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 660 gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 660

gagtgcatac agggtggcgg tggctcgggc ggtggtgggt cgggtggcgg cggatctggt 720 gagtgcatac agggtggcgg tggctcgggc ggtggtgggt cgggtggcgg cggatctggt 720

ggcggtggct cgttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 780 ggcggtggct cgttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 780

ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagc 828 ctagtaacag tggcctttat tattttctgg gtgaggagta agaggago 828

<210> 80 <210> 80 <211> 885 <211> 885 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 4 (Her1 truncated design 4) <223> /note="EGFR truncated design 4 (Her1 truncated design 4) (HER1t4)" (HER1t4)'

<400> 80 <400> 80 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 cgcaaaattgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 Page 40 Page 40

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 300

ggctgctggg gcccggagcc cagggactgo gtctcttgcc ggaatgtcag ccgaggcagg ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600 600

gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720 720

cggggaccag acaactgtat ccagggcgga ggcggaagcg gaggcggagg ctccggcgga 780 cggggaccag acaactgtat ccagggcgga ggcggaagcg gaggcggagg ctccggcgga 780

ggcggaagct tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta tagcttgcta ggcggaagct tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta tagcttgcta 840 840

gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagc 885 gtaacagtgg cctttattat tttctgggtg aggagtaaga ggago 885

<210> 81 <210> 81 <211> 924 <211> 924 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note= 'Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 5 (Her1 truncated design 5) <223> /note="EGFR truncated design 5 (Her1 truncated design 5) (HER1t5)" (HER1t5)' "

<400> 81 <400> 81 cgcaaaatt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 60

acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 120

Page 41 Page 41

50471‐706_601_SL.TXT 50471-706_601_SL.TXT aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggad caagcaacat 300

agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 agaggtgaaa acagctgcaa ggccacaggo caggtctgcc atgccttgtg ctcccccgag 540

ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600 ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600

gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720 gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720

accggcggag gcggaagcgg aggcggaggc tccggcggag gcggaagctt ttgggtgctg 840 accggcggag gcggaagcgg aggcggaggo tccggcggag gcggaagctt ttgggtgctg 840

gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 900 gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 900

ttctgggtga ggagtaagag gagc 924 ttctgggtga ggagtaagag gagc 924

<210> 82 <210> 82 <211> 984 <211> 984 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 6 (Her1 truncated design 6) <223> /note="EGFR truncated design 6 (Her1 truncated design 6) (HER1t6)" (HER1t6)'

<400> 82 <400> 82 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 cgcaaaatt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360 ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360 Page 42 Page 42

50471‐706_601_SL.TXT 50471-706_601_SL.TXT aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420 420 acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480 480 agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 540 ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600 600 gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 660 660 gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720 720

cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag 780 780 acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 840 840 ggccatgtgt gccacctggg cggaggcgga agcggaggcg gaggctcctt ttgggtgctg ggccatgtgt gccacctggg cggaggcgga agcggaggcg gaggctcctt ttgggtgctg 900 900 gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 960 960

ttctgggtga ggagtaagag gagc 984 ttctgggtga ggagtaagag gagc 984

<210> 83 <210> 83 <211> 1032 <211> 1032 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> /note="EGFR source <223> truncated design 7 (Her1 truncated design 7) <223> /note="EGFR truncated design 7 (Her1 truncated design 7) (HER1t7)" (HER1t7) "

<400> 83 <400> 83 cgcaaaattgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 60 acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 120 gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180 180 attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240 240

aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggad caagcaacat aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 300 ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360 360

Page 43 Page 43

50471‐706_601_SL.TXT 50471-706_601_SL.TXT aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420 aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420

acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 840 acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 840

ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt 900 ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt 900

gaaggctgtc caggtggcgg tggcggcgga tctttttggg tgctggtggt ggttggtgga 960 gaaggctgtc caggtggcgg tggcggcgga tctttttggg tgctggtggt ggttggtgga 960

gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1020 gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1020

aagaggagct aa 1032 aagaggagct aa 1032

<210> 84 <210> 84 <211> 726 <211> 726 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 8 (Her1 truncated design 8) <223> /note="EGFR truncated design 8 (Her1 truncated design 8) (HER1t8)" (HER1t8) "

<400> 84 <400> 84 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 cgcaaaatt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

Page 44 Page 44

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420 420 acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480 480 agaggtgaaa acagctgcaa ggccacaggo caggtctgcc atgccttgtg ctcccccgag agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 540 ggctgctggg gcccggagcc cagggactgc gtctctggtg gcggtggctc gggcggtggt ggctgctggg gcccggagcc cagggactgc gtctctggtg gcggtggctc gggcggtggt 600 600 gggtcgggtg gcggcggatc tggtggcggt ggctcggaga taacactcat tatttttggg gggtcgggtg gcggcggatc tggtggcggt ggctcggaga taacactcat tatttttggg 660 660 gtgatggctg gtgttattgg aacgatcctc ttaatttctt acggtattcg ccgaggaggt gtgatggctg gtgttattgg aacgatcctc ttaatttctt acggtattcg ccgaggaggt 720 720

ggaagc 726 ggaagc 726

<210> 85 <210> 85 <211> 717 <211> 717 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> <223> source /note="EGFR truncated design 9 (Her1 truncated design 9) <223> /note="EGFR truncated design 9 (Her1 truncated design 9) (HER1t9)" (HER1t9)' "

<400> 85 <400> 85 cgcaaaattgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 60 acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 120 gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180 180 attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240 240 aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggad caagcaacat aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 300 ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360 360 aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420 420 acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480 480 agaggtgaaa acagctgcaa ggccacaggc caggtctgco atgccttgtg ctcccccgag agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 540 ggctgctggg gcccggagcc cagggactgc gtctctggtg gcggtggctc gggcggtggt ggctgctggg gcccggagcc cagggactgc gtctctggtg gcggtggctc gggcggtggt 600 600

Page 45 Page 45

50471‐706_601_SL.TXT 50471-706_601_SL.TXT gggtcgggtg gcggcggatc tggtggcggt ggctcgataa cactcattat ttttggggtg 660 gggtcgggtg gcggcggatc tggtggcggt ggctcgataa cactcattat ttttggggtg 660

atggctggtg ttattggaac gatcctctta atttcttacg gtattggagg tggaagc 717 atggctggtg ttattggaac gatcctctta atttcttacg gtattggagg tggaagc 717

<210> 86 <210> 86 <211> 717 <211> 717 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 10 (Her1 truncated design 10) <223> /note="EGFR truncated design 10 (Her1 truncated design 10) (HER1t10)" (HER101)

<400> 86 <400> 86 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 cgcaaaattgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggad caagcaacat 300

ggctgctggg gcccggagcc cagggactgc gtctctggtg gcggtggctc gggcggtggt 600 ggctgctggg gcccggagcc cagggactgc gtctctggtg gcggtggctc gggcggtggt 600

gggtcgggtg gcggcggatc tggtggcggt ggctcgataa cactcattat ttttggggtg 660 gggtcgggtg gcggcggatc tggtggcggt ggctcgataa cactcattat ttttggggtg 660

atggctggtg ttattggaac gatcctctta gccctgctca tctggggagg tggaagc 717 atggctggtg ttattggaac gatcctctta gccctgctca tctggggagg tggaagc 717

<210> 87 <210> 87 <211> 711 <211> 711 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> Page 46 Page 46

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 11 (Her1 truncated design 11) <223> /note="EGFR truncated design 11 (Her1 truncated design 11) (HER1t11)" (HER1t11)"

<400> 87 <400> 87 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 cgcaaaatt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 agaggtgaaa acagctgcaa ggccacaggc caggtctgco atgccttgtg ctcccccgag 540

gggtcgggtg gcggcggatc tggtggcggt ggctcgctct gctacctgct ggatggaatc 660 gggtcgggtg gcggcggatc tggtggcggt ggctcgctct gctacctgct ggatggaatc 660

ctcttcatct atggtgtcat tctcactgcc ttgttcctgg gaggtggaag c 711 ctcttcatct atggtgtcat tctcactgcc ttgttcctgg gaggtggaag C 711

<210> 88 <210> 88 <211> 891 <211> 891 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="FL CD20" <223> /note="FL CD20"

<400> 88 <400> 88 atgacaacac ccagaaattc agtaaatggg actttcccgg cagagccaat gaaaggccct 60 atgacaacao ccagaaattc agtaaatggg actttcccgg cagagccaat gaaaggccct 60

Page 47 Page 47

50471‐706_601_SL.TXT 50471-706_601_SL.TXT attgctatgc aatctggtcc aaaaccacto ttcaggagga tgtcttcact ggtgggcccc attgctatgc aatctggtcc aaaaccactc ttcaggagga tgtcttcact ggtgggcccc 120 120

acgcaaagct tcttcatgag ggaatctaag actttggggg ctgtccagat tatgaatggg acgcaaagct tcttcatgag ggaatctaag actttggggg ctgtccagat tatgaatggg 180 180

ctcttccaca ttgccctggg gggtcttctg atgatcccag cagggatcta tgcacccato ctcttccaca ttgccctggg gggtcttctg atgatcccag cagggatcta tgcacccatc 240 240

tgtgtgactg tgtggtaccc tctctgggga ggcattatgt atattattto cggatcacto tgtgtgactg tgtggtaccc tctctgggga ggcattatgt atattatttc cggatcactc 300 300

ctggcagcaa cggagaaaaa ctccaggaag tgtttggtca aaggaaaaat gataatgaat ctggcagcaa cggagaaaaa ctccaggaag tgtttggtca aaggaaaaat gataatgaat 360 360

tcattgagcc tctttgctgc catttctgga atgattcttt caatcatgga catacttaat tcattgagcc tctttgctgc catttctgga atgattcttt caatcatgga catacttaat 420 420

attaaaattt cccatttttt aaaaatggag agtctgaatt ttattagago tcacacacca attaaaattt cccatttttt aaaaatggag agtctgaatt ttattagagc tcacacacca 480 480

tatattaaca tatacaactg tgaaccagct aatccctctg agaaaaactc cccatctacc tatattaaca tatacaactg tgaaccagct aatccctctg agaaaaactc cccatctacc 540 540

caatactgtt acagcataca atctctgttc ttgggcattt tgtcagtgat gctgatcttt caatactgtt acagcataca atctctgttc ttgggcattt tgtcagtgat gctgatcttt 600 600

gccttcttcc aggaacttgt aatagctggo atcgttgaga atgaatggaa aagaacgtgo gccttcttcc aggaacttgt aatagctggc atcgttgaga atgaatggaa aagaacgtgc 660 660

tccagaccca aatctaacat agttctcctg tcagcagaag aaaaaaaaga acagactatt tccagaccca aatctaacat agttctcctg tcagcagaag aaaaaaaaga acagactatt 720 720

gaaataaaag aagaagtggt tgggctaact gaaacatctt cccaaccaaa gaatgaagaa gaaataaaag aagaagtggt tgggctaact gaaacatctt cccaaccaaa gaatgaagaa 780 780

gacattgaaa ttattccaat ccaagaagag gaagaagaag aaacagagad gaactttcca gacattgaaa ttattccaat ccaagaagag gaagaagaag aaacagagac gaactttcca 840 840

gaacctcccc aagatcagga atcctcacca atagaaaatg acagctctcc t 891 gaacctcccc aagatcagga atcctcacca atagaaaatg acagctctco t 891

<210> 89 <210> 89 <211> 789 <211> 789 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Truncated CD20 design 1 (CD20t1) [CD20(M1-E263]" " <223> /note="Truncated CD20 design 1 (CD20t1) [CD20(M1‐E263]"

<400> 89 <400> 89 atgaccacac cacggaactc tgtgaatggc accttcccag cagagccaat gaagggacca atgaccacac cacggaactc tgtgaatggc accttcccag cagagccaat gaagggacca 60 60

atcgcaatgc agagcggacc caagcctctg tttcggagaa tgagctccct ggtgggccca atcgcaatgc agagcggacc caagcctctg tttcggagaa tgagctccct ggtgggccca 120 120

acccagtcct tctttatgag agagtctaag acactgggcg ccgtgcagat catgaacgga acccagtcct tctttatgag agagtctaag acactgggcg ccgtgcagat catgaacgga 180 180

ctgttccaca tcgccctggg aggactgctg atgatcccag ccggcatcta cgcccctato ctgttccaca tcgccctggg aggactgctg atgatcccag ccggcatcta cgcccctatc 240 240

Page 48 Page 48

50471‐706_601_SL.TXT 50471-706_601_SL.TXT tgcgtgaccg tgtggtaccc tctgtggggo ggcatcatgt atatcatctc cggctctctg tgcgtgaccg tgtggtaccc tctgtggggc ggcatcatgt atatcatctc cggctctctg 300 300

ctggccgcca cagagaagaa cagcaggaag tgtctggtga agggcaagat gatcatgaat ctggccgcca cagagaagaa cagcaggaag tgtctggtga agggcaagat gatcatgaat 360 360

agcctgtccc tgtttgccgc catctctggc atgatcctga gcatcatgga catcctgaac agcctgtccc tgtttgccgc catctctggc atgatcctga gcatcatgga catcctgaac 420 420 atcaagatca gccacttcct gaagatggag agcctgaact tcatcagage ccacacccct atcaagatca gccacttcct gaagatggag agcctgaact tcatcagagc ccacacccct 480 480 tacatcaaca tctataattg cgagcctgcc aacccatccg agaagaattc tccaagcaca tacatcaaca tctataattg cgagcctgcc aacccatccg agaagaattc tccaagcaca 540 540

cagtactgtt attccatcca gtctctgttc ctgggcatcc tgtctgtgat gctgatcttt cagtactgtt attccatcca gtctctgttc ctgggcatcc tgtctgtgat gctgatcttt 600 600

gccttctttc aggagctggt catcgccggc atcgtggaga acgagtggaa gaggacctgo gccttctttc aggagctggt catcgccggc atcgtggaga acgagtggaa gaggacctgc 660 660 agccgcccca agtccaatat cgtgctgctg tccgccgagg agaagaagga gcagacaatc agccgcccca agtccaatat cgtgctgctg tccgccgagg agaagaagga gcagacaatc 720 720 gagatcaagg aggaggtggt gggcctgacc gagacatcta gccagcctaa gaatgaggag gagatcaagg aggaggtggt gggcctgacc gagacatcta gccagcctaa gaatgaggag 780 780

gatatcgag 789 gatatcgag 789

<210> 90 <210> 90 <211> 342 <211> 342 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Interleukin‐15" <223> /note="Interleukin-15"

<400> 90 <400> 90 aactgggtga atgtgatcag cgacctgaag aagatcgagg atctgatcca gagcatgcad aactgggtga atgtgatcag cgacctgaag aagatcgagg atctgatcca gagcatgcac 60 60 attgatgcca ccctgtacac agaatctgat gtgcacccta gctgtaaagt gaccgccatg attgatgcca ccctgtacac agaatctgat gtgcacccta gctgtaaagt gaccgccatg 120 120

aagtgttttc tgctggagct gcaggtgatt tctctggaaa gcggagatgc ctctatccac aagtgttttc tgctggagct gcaggtgatt tctctggaaa gcggagatgc ctctatccac 180 180

gacacagtgg agaatctgat catcctggcc aacaatagcc tgagcagcaa tggcaatgtg gacacagtgg agaatctgat catcctggcc aacaatagcc tgagcagcaa tggcaatgtg 240 240

acagagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagtttctg acagagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagtttctg 300 300

cagagctttg tgcacatcgt gcagatgttc atcaatacaa gc cagagctttg tgcacatcgt gcagatgttc atcaatacaa gc 342 342

<210> 91 <210> 91 <211> 711 <211> 711 <212> DNA <212> DNA Page 49 Page 49

<220> <220> <221> source <221> source <223> /note="Interleukin‐15 receptor alpha" <223> /note="Interleukin-15 receptor alpha"

<400> 91 <400> 91 attacatgcc ctcctccaat gtctgtggag cacgccgata tttgggtgaa gtcctacagc 60 attacatgcc ctcctccaat gtctgtggag cacgccgata tttgggtgaa gtcctacago 60

ctgtacagca gagagagata catctgcaac agcggcttta agagaaaggc cggcacctct 120 ctgtacagca gagagagata catctgcaac agcggcttta agagaaaggc cggcacctct 120

tctctgacag agtgcgtgct gaataaggcc acaaatgtgg cccactggac aacacctagc 180 tctctgacag agtgcgtgct gaataaggcc acaaatgtgg cccactggad aacacctago 180

ctgaagtgca ttagagatcc tgccctggtc caccagaggc ctgcccctcc atctacagtg 240 ctgaagtgca ttagagatcc tgccctggtc caccagaggc ctgcccctcc atctacagtg 240

acaacagccg gagtgacacc tcagcctgaa tctctgagcc cttctggaaa agaacctgcc 300 acaacagccg gagtgacacc tcagcctgaa tctctgagcc cttctggaaa agaacctgcc 300

gccagctctc ctagctctaa taataccgcc gccacaacag ccgccattgt gcctggatct 360 gccagctctc ctagctctaa taataccgcc gccacaacag ccgccattgt gcctggatct 360

cagctgatgc ctagcaagtc tcctagcaca ggcacaacag agatcagcag ccacgaatct 420 cagctgatgc ctagcaagto tcctagcaca ggcacaacag agatcagcag ccacgaatct 420

tctcacggaa caccttctca gaccaccgcc aagaattggg agctgacagc ctctgcctct 480 tctcacggaa caccttctca gaccaccgcc aagaattggg agctgacago ctctgcctct 480

caccagcctc caggagtgta tcctcagggc cactctgata caacagtggc catcagcaca 540 caccagcctc caggagtgta tcctcagggc cactctgata caacagtggc catcagcaca 540

tctacagtgc tgctgtgtgg actgtctgcc gtgtctctgc tggcctgtta cctgaagtct 600 tctacagtgo tgctgtgtgg actgtctgcc gtgtctctgc tggcctgtta cctgaagtct 600

agacagacac ctcctctggc ctctgtggag atggaggcca tggaagccct gcctgtgaca 660 agacagacac ctcctctggc ctctgtggag atggaggcca tggaagccct gcctgtgaca 660

tggggaacaa gcagcagaga tgaggacctg gagaattgtt ctcaccacct g 711 tggggaacaa gcagcagaga tgaggacctg gagaattgtt ctcaccacct g 711

<210> 92 <210> 92 <211> 1185 <211> 1185 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Membrane bound Interleukin‐15 with signal peptide" <223> /note="Membrane bound Interleukin-15 with signal peptide"

<400> 92 <400> 92 Page 50 Page 50

50471‐706_601_SL.TXT 50471-706_601_SL.TXT atggattgga cctggattct gtttctggtg gccgctgcca caagagtgca cagcaactgg 60 atggattgga cctggattct gtttctggtg gccgctgcca caagagtgca cagcaactgg 60

gtgaatgtga tcagcgacct gaagaagatc gaggatctga tccagagcat gcacattgat 120 gtgaatgtga tcagcgacct gaagaagatc gaggatctga tccagagcat gcacattgat 120

gccaccctgt acacagaatc tgatgtgcac cctagctgta aagtgaccgc catgaagtgt 180 gccaccctgt acacagaato tgatgtgcac cctagctgta aagtgaccgc catgaagtgt 180

tttctgctgg agctgcaggt gatttctctg gaaagcggag atgcctctat ccacgacaca 240 tttctgctgg agctgcaggt gatttctctg gaaagcggag atgcctctat ccacgacaca 240

gtggagaatc tgatcatcct ggccaacaat agcctgagca gcaatggcaa tgtgacagag 300 gtggagaatc tgatcatcct ggccaacaat agcctgagca gcaatggcaa tgtgacagag 300

tctggctgta aggagtgtga ggagctggag gagaagaaca tcaaggagtt tctgcagagc 360 tctggctgta aggagtgtga ggagctggag gagaagaaca tcaaggagtt tctgcagago 360

tttgtgcaca tcgtgcagat gttcatcaat acaagctctg gcggaggatc tggaggaggc 420 tttgtgcaca tcgtgcagat gttcatcaat acaagctctg gcggaggatc tggaggaggo 420

ggatctggag gaggaggcag tggaggcgga ggatctggcg gaggatctct gcagattaca 480 ggatctggag gaggaggcag tggaggcgga ggatctggcg gaggatctct gcagattaca 480

tgccctcctc caatgtctgt ggagcacgcc gatatttggg tgaagtccta cagcctgtac 540 tgcccctcctc caatgtctgt ggagcacgcc gatatttggg tgaagtccta cagcctgtac 540

agcagagaga gatacatctg caacagcggc tttaagagaa aggccggcac ctcttctctg 600 agcagagaga gatacatctg caacagcggc tttaagagaa aggccggcac ctcttctctg 600

acagagtgcg tgctgaataa ggccacaaat gtggcccact ggacaacacc tagcctgaag 660 acagagtgcg tgctgaataa ggccacaaat gtggcccact ggacaacacc tagcctgaag 660

tgcattagag atcctgccct ggtccaccag aggcctgccc ctccatctac agtgacaaca 720 tgcattagag atcctgccct ggtccaccag aggcctgccc ctccatctac agtgacaaca 720

gccggagtga cacctcagcc tgaatctctg agcccttctg gaaaagaacc tgccgccagc 780 gccggagtga cacctcagcc tgaatctctg agcccttctg gaaaagaacc tgccgccago 780

tctcctagct ctaataatac cgccgccaca acagccgcca ttgtgcctgg atctcagctg 840 tctcctagct ctaataatac cgccgccaca acagccgcca ttgtgcctgg atctcagctg 840

atgcctagca agtctcctag cacaggcaca acagagatca gcagccacga atcttctcac 900 atgcctagca agtctcctag cacaggcaca acagagatca gcagccacga atcttctcac 900

ggaacacctt ctcagaccac cgccaagaat tgggagctga cagcctctgc ctctcaccag 960 ggaacacctt ctcagaccad cgccaagaat tgggagctga cagcctctgc ctctcaccag 960

cctccaggag tgtatcctca gggccactct gatacaacag tggccatcag cacatctaca 1020 cctccaggag tgtatcctca gggccactct gatacaacag tggccatcag cacatctaca 1020

gtgctgctgt gtggactgtc tgccgtgtct ctgctggcct gttacctgaa gtctagacag 1080 gtgctgctgt gtggactgtc tgccgtgtct ctgctggcct gttacctgaa gtctagacag 1080

acacctcctc tggcctctgt ggagatggag gccatggaag ccctgcctgt gacatgggga 1140 acacctcctc tggcctctgt ggagatggag gccatggaag ccctgcctgt gacatgggga 1140

acaagcagca gagatgagga cctggagaat tgttctcacc acctg 1185 acaagcagca gagatgagga cctggagaat tgttctcacc acctg 1185

<210> 93 <210> 93 <211> 1605 <211> 1605 <212> DNA <212> DNA <213> Mus sp. <213> Mus sp.

<220> <220> <221> source <221> source <223> /note="Murine single chain IL‐12 (p40‐linker‐p35)" <223> /note="Murine single chain IL-12 (p40-linker-p35)" "

<400> 93 <400> 93 atgtgtcctc agaagctaac catctcctgg tttgccatcg ttttgctggt gtctccactc 60 atgtgtcctc agaagctaac catctcctgg tttgccatcg ttttgctggt gtctccactc 60 Page 51 Page 51

50471‐706_601_SL.TXT 1x1'7S

atggccatgt gggagctgga gaaagacgtt tatgttgtag aggtggactg gactcccgat 120

The gcccctggag aaacagtgaa cctcacctgt gacacgcctg aagaagatga catcacctgg 180 08T

acctcagacc agagacatgg agtcataggc tctggaaaga ccctgaccat cactgtcaaa 240 DATE

gagtttctag atgctggcca gtacacctgc cacaaaggag gcgagactct gagccactca 300 00E

catctgctgc tccacaagaa ggaaaatgga atttggtcca ctgaaattct gaagaacttc 360 09E

aaaaacaaga ctttcctgaa gtgtgaagca ccaaattaca gcggccggtt cacgtgctca 420

7 tggctggtgc aaagaaacat ggacttgaag ttcaacatca agagcagtag cagttcccct 480 08/

e gactctcggg cagtgacatg tggaatggcg tctctgagcg ccgagaaggt cacactggac 540

cagagagact atgagaagta ttcagtgtcc tgccaggagg atgtcacctg cccaactgcc 600 009

gaggagaccc tgcccattga actggcgttg gaagcacggc agcagaacaa gtatgagaac 660 099

tacagcacca gcttcttcat cagggacatc atcaaaccag acccgcccaa gaacttgcag 720 02L

atgaagcctt tgaagaactc acaggtggag gtcagctggg agtaccctga ctcctggagc 780 08L

actccccatt cctacttctc cctcaagttc tttgtgagaa tccagcgcaa gaaagaaaag 840

atgaaggaga cagaggaggg gtgtaaccag aaaggtgcgt tcctcgtaga gaagacatct 900 006

accgaagtcc aatgcaaagg cgggaatgtc tgcgtgcaag ctcaggatcg ctattacaat 960 096

e tcctcatgca gcaagtgggc atgtgttccc tgccgcgtcc gatccggtgg cggtggcggc 1020 020T

ggatctaggg tcattccagt ctctggacct gccaggtgtc ttagccagtc ccgaaacctg 1080 080T

ctgaagacca cagatgacat ggtgaagacg gccagagaaa aactgaaaca ttattcctgc 1140

actgctgaag acattgacca tgaagacatc acacgggacc aaaccagcac attgaagacc 1200 present tgtttaccac tggaactaca caagaacgag agttgcctgg ctactagaga gacttcttcc 1260 092T

acaacaagag ggagctgcct gcccccacag aagaccagct tgatgatgac cctgtgcctt 1320 OZET

ggtagcatct atgaggactt gaagatgtac cagacagagt tccaggccat caacgcagca 1380 08ET

the cttcagaatc acaaccatca gcagatcatt ctagacaagg gcatgttggt ggccatcgac 1440

gagctgatgc agtctctgaa tcataatggc gagactctgc gccagaaacc tcctgtggga 1500 00ST

gaagcagacc cttacagagt gaaaatgaag ctctgcatcc tgcttcacgc cttcagcacc 1560 09ST

cgcgtcgtga ccatcaacag ggtgatgggc tatctgagca gcgcc 1605 SO9T

Page 52 25 aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<210> 94 <210> 94 <211> 1596 <211> 1596 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<220> <220> <221> source <221> source <223> /note="Human single chain IL‐12 (p40‐linker‐p35)" <223> /note="Human single chain IL-12 (p40-linker-p35)" "

<400> 94 <400> 94 atgtgtcacc agcagttggt catctcttgg ttcagcctgg tttttctggc atctcccctc 60 atgtgtcaco agcagttggt catctcttgg ttcagcctgg tttttctggc atctcccctc 60

gtggccatct gggaactgaa gaaagatgtt tatgtcgtag aattggattg gtatcccgac 120 gtggccatct gggaactgaa gaaagatgtt tatgtcgtag aattggattg gtatcccgac 120

gcccctggag aaatggtggt cctgacatgt gacacccctg aagaagatgg tatcacctgg 180 gccccctggag aaatggtggt cctgacatgt gacacccctg aagaagatgg tatcacctgg 180

accttggacc agagcagtga ggtcttaggc tctggcaaga ccctgaccat ccaagtcaaa 240 accttggacc agagcagtga ggtcttaggc tctggcaaga ccctgaccat ccaagtcaaa 240

gagtttggag atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg 300 gagtttggag atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg 300

ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgacattct gaaggaccag 360 ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgacattct gaaggaccag 360

aaagaaccca agaataagac ctttctaaga tgcgaggcca agaattattc tggacgtttc 420 aaagaaccca agaataagac ctttctaaga tgcgaggcca agaattatto tggacgtttc 420

acctgctggt ggctgacgac aatcagtact gatttgacat tcagtgtcaa aagcagcaga 480 acctgctggt ggctgacgac aatcagtact gatttgacat tcagtgtcaa aagcagcaga 480

ggctcttctg acccccaagg ggtgacgtgc ggagctgcta cactcagcgc cgagagagtc 540 ggctcttctg acccccaagg ggtgacgtgc ggagctgcta cactcagcgc cgagagagto 540

agaggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca 600 agaggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca 600

gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660 gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660

gaaaactaca ccagcagctt cttcatcagg gacatcatca aacctgaccc acccaagaac 720 gaaaactaca ccagcagctt cttcatcagg gacatcatca aacctgaccc acccaagaac 720

ttgcagctga agcccctgaa gaacagcaga caggtggagg tcagctggga gtaccctgac 780 ttgcagctga agcccctgaa gaacagcaga caggtggagg tcagctggga gtaccctgac 780

acctggagta ctccacattc ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840 acctggagta ctccacatto ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840

agcaagagag aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc 900 agcaagagag aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc 900

cgcaaaaatg ccagcattag cgtgcgggcc caggaccgct actatagctc atcttggagc 960 cgcaaaaatg ccagcattag cgtgcgggcc caggaccgct actatagctc atcttggago 960

gaatgggcat ctgtgccctg ctccggtggc ggtggcggcg gatctagaaa cctccccgtg 1020 gaatgggcat ctgtgccctg ctccggtggc ggtggcggcg gatctagaaa cctccccgtg 1020

gccactccag acccaggaat gttcccatgc cttcaccaca gccagaacct gctgagggcc 1080 gccactccag acccaggaat gttcccatgc cttcaccaca gccagaacct gctgagggcc 1080

gtcagcaaca tgctccagaa ggccagacaa actctagaat tttacccttg cacttctgaa 1140 gtcagcaaca tgctccagaa ggccagacaa actctagaat tttacccttg cacttctgaa 1140

gagattgatc atgaagatat cacaaaagat aaaaccagca cagtggaggc ctgtttacca 1200 gagattgatc atgaagatat cacaaaagat aaaaccagca cagtggaggo ctgtttacca 1200

Page 53 Page 53

50471‐706_601_SL.TXT 50471-706_601_SL.TXT ttggaattaa ccaagaatga gagttgccta aattccagag agacctcttt cataactaat ttggaattaa ccaagaatga gagttgccta aattccagag agacctcttt cataactaat 1260 1260

gggagttgcc tggcctccag aaagacctct tttatgatgg ccctgtgcct tagtagtatt gggagttgcc tggcctccag aaagacctct tttatgatgg ccctgtgcct tagtagtatt 1320 1320

tatgaagact tgaagatgta ccaggtggag ttcaagacca tgaatgcaaa gctgctgatg tatgaagact tgaagatgta ccaggtggag ttcaagacca tgaatgcaaa gctgctgatg 1380 1380

gaccccaaga ggcagatctt tctagatcaa aacatgctgg cagttattga tgagctgatg gaccccaaga ggcagatctt tctagatcaa aacatgctgg cagttattga tgagctgatg 1440 1440

caggccctga atttcaacag tgagactgtg ccacaaaaat cctcccttga agaaccggat caggccctga atttcaacag tgagactgtg ccacaaaaat cctcccttga agaaccggat 1500 1500

ttttataaaa ctaaaatcaa gctctgcata cttcttcatg ctttcagaat cagagcagtg ttttataaaa ctaaaatcaa gctctgcata cttcttcatg ctttcagaat cagagcagtg 1560 1560

actattgata gagtgatgag ctatctgaat gcttcc 1596 actattgata gagtgatgag ctatctgaat gcttcc 1596

<210> 95 <210> 95 <211> 6684 <211> 6684 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<220> <220> <221> source <221> source <223> /note="Human interleukin-2 (IL-2) gene and 5'-flanking <223> /note="Human interleukin‐2 (IL‐2) gene and 5'‐flanking region" region"

<400> 95 <400> 95 agtggttttt ggagtcagta cattctcttt tcaaatcctt ctctgcccct tactggcaat agtggttttt ggagtcagta cattctcttt tcaaatcctt ctctgcccct tactggcaat 60 60

aagggctgag tgacctagag gcaaattact taacttctct gagcctcagt tttctaatct aagggctgag tgacctagag gcaaattact taacttctct gagcctcagt tttctaatct 120 120

gcaaaatagg agccatcact tcacaagtct gtaagactta tattagacta agtgcctgcc gcaaaatagg agccatcact tcacaagtct gtaagactta tattagacta agtgcctgcc 180 180

tgtacactgt tctctttttc tctctttcta tatacctgaa ggcattatag tgctagatgt tgtacactgt tctctttttc tctctttcta tatacctgaa ggcattatag tgctagatgt 240 240

ctgtttaaag accagacaat attgtcttaa aaaaacaaac aaaaacacag acaataccat ctgtttaaag accagacaat attgtcttaa aaaaacaaac aaaaacacag acaataccat 300 300

ctttaaaaaa aaaaaaaaag tccaggtaag aaataaataa ggccatagaa tggaagcttt ctttaaaaaa aaaaaaaaag tccaggtaag aaataaataa ggccatagaa tggaagcttt 360 360

acaaggactc tctttgagac aggatctcct caagtgtccc caggttaaat tagaagtata acaaggactc tctttgagac aggatctcct caagtgtccc caggttaaat tagaagtata 420 420 tatccgtaca attgttcagc cagtttgtgo actgtactga ggatgaatga acacctatco tatccgtaca attgttcagc cagtttgtgc actgtactga ggatgaatga acacctatcc 480 480 taaatatcct agtcttctga ctaaaaacaa gatcatattt cataacgatt attgttacat taaatatcct agtcttctga ctaaaaacaa gatcatattt cataacgatt attgttacat 540 540

tcatagtgtc ccaggtgatt tagaggataa ataaaaatcc attaaagagg taaagacata tcatagtgtc ccaggtgatt tagaggataa ataaaaatcc attaaagagg taaagacata 600 600

aaaacgagaa acatggactg gtttacacat aacacataca aagtctatta taaaactago aaaacgagaa acatggactg gtttacacat aacacataca aagtctatta taaaactagc 660 660

atcagtatco ttgaatcgaa acctttttct gagtatttaa caatcgcaco ctttaaaaaa atcagtatcc ttgaatcgaa acctttttct gagtatttaa caatcgcacc ctttaaaaaa 720 720

tgtacataga cattaagaga cttaaacaga tatataatca ttttaaatta aaatagcgtt tgtacataga cattaagaga cttaaacaga tatataatca ttttaaatta aaatagcgtt 780 780

Page 54 Page 54

50471‐706_601_SL.TXT 50471-706_601_SL.TXT aaacagtacc tcaagctcaa taagcatttt aagtattcta atcttagtat ttctctagct 840 aaacagtacc tcaagctcaa taagcatttt aagtattcta atcttagtat ttctctagct 840

gacatgtaag aagcaatcta tcttattgta tgcaattagc tctttgtgtg gataaaaagg 900 gacatgtaag aagcaatcta tcttattgta tgcaattagc tctttgtgtg gataaaaagg 900

taaaaccatt ctgaaacagg aaaccaatac acttcctgtt taatcaacaa atctaaacat 960 taaaaccatt ctgaaacagg aaaccaatac acttcctgtt taatcaacaa atctaaacat 960

ttattctttt catctgttta ctcttgctct tgtccaccac aatatgctat tcacatgttc 1020 ttattctttt catctgttta ctcttgctct tgtccaccac aatatgctat tcacatgtto 1020

agtgtagttt tatgacaaag aaaattttct gagttacttt tgtatcccca cccccttaaa 1080 agtgtagttt tatgacaaag aaaattttct gagttacttt tgtatcccca cccccttaaa 1080

gaaaggagga aaaactgttt catacagaag gcgttaattg catgaattag agctatcacc 1140 gaaaggagga aaaactgttt catacagaag gcgttaattg catgaattag agctatcacc 1140

taagtgtggg ctaatgtaac aaagagggat ttcacctaca tccattcagt cagtctttgg 1200 taagtgtggg ctaatgtaac aaagagggat ttcacctaca tccattcagt cagtctttgg 1200

gggtttaaag aaattccaaa gagtcatcag aagaggaaaa atgaaggtaa tgttttttca 1260 gggtttaaag aaattccaaa gagtcatcag aagaggaaaa atgaaggtaa tgttttttca 1260

gactggtaaa gtctttgaaa atatgtgtaa tatgtaaaac attttgacac ccccataata 1320 gactggtaaa gtctttgaaa atatgtgtaa tatgtaaaac attttgacac ccccataata 1320

tttttccaga attaacagta taaattgcat ctcttgttca agagttccct atcactcttt 1380 tttttccaga attaacagta taaattgcat ctcttgttca agagttccct atcactcttt 1380

aatcactact cacagtaacc tcaactcctg ccacaatgta caggatgcaa ctcctgtctt 1440 aatcactact cacagtaacc tcaactcctg ccacaatgta caggatgcaa ctcctgtctt 1440

gcattgcact aagtcttgca cttgtcacaa acagtgcacc tacttcaagt tctacaaaga 1500 gcattgcact aagtcttgca cttgtcacaa acagtgcacc tacttcaagt tctacaaaga 1500

aaacacagct acaactggag catttactgc tggatttaca gatgattttg aatggaatta 1560 aaacacagct acaactggag catttactgc tggatttaca gatgattttg aatggaatta 1560

atgtaagtat atttcctttc ttactaaaat tattacattt agtaatctag ctggagatca 1620 atgtaagtat atttcctttc ttactaaaat tattacattt agtaatctag ctggagatca 1620

tttcttaata acaatgcatt atactttctt agaattacaa gaatcccaaa ctcaccagga 1680 tttcttaata acaatgcatt atactttctt agaattacaa gaatcccaaa ctcaccagga 1680

tgctcacatt taagttttac atgcccaaga aggtaagtac aatattttat gttcaatttc 1740 tgctcacatt taagttttac atgcccaaga aggtaagtac aatattttat gttcaatttc 1740

tgttttaata aaattcaaag taatatgaaa atttgcacag atgggactaa tagcagctca 1800 tgttttaata aaattcaaag taatatgaaa atttgcacag atgggactaa tagcagctca 1800

tctgaggtaa agagtaactt taatttgttt ttttgaaaac ccaagtttga taatgaagcc 1860 tctgaggtaa agagtaactt taatttgttt ttttgaaaac ccaagtttga taatgaagcc 1860

tctattaaaa cagttttacc tatattttta atatatattt gtgtgttggt gggggtggga 1920 tctattaaaa cagttttacc tatattttta atatatattt gtgtgttggt gggggtggga 1920

gaaaacataa aaataatatt ctctcacttt atcgataaga caattctaaa caaaaatgtt 1980 gaaaacataa aaataatatt ctctcacttt atcgataaga caattctaaa caaaaatgtt 1980

catttatggt ttcatttaaa aatgtaaaac tctaaaatat ttgattatgt cattttagta 2040 catttatggt ttcatttaaa aatgtaaaac tctaaaatat ttgattatgt cattttagta 2040

tgtaaaatac caaaatctat ttccaaggag cccactttta aaaatctttt cttgttttag 2100 tgtaaaatac caaaatctat ttccaaggag cccactttta aaaatctttt cttgttttag 2100

gaaaggtttc taagtgagag gcagcataac actaatagca cagagtctgg ggccagatat 2160 gaaaggtttc taagtgagag gcagcataac actaatagca cagagtctgg ggccagatat 2160

ctgaagtgaa atctcagctc tgccatgtcc tagctttcat gatctttggc aaattaccta 2220 ctgaagtgaa atctcagctc tgccatgtcc tagctttcat gatctttggc aaattaccta 2220

ctctgtttgt gattcagttt catgtctact taaatgaata actgtatata cttaatatgg 2280 ctctgtttgt gattcagttt catgtctact taaatgaata actgtatata cttaatatgg 2280

ctttgtgaga attagtaagt aaatgtaaag cactcagaac cgtgtctggc ataaggtaaa 2340 ctttgtgaga attagtaagt aaatgtaaag cactcagaac cgtgtctggc ataaggtaaa 2340

Page 55 Page 55

50471‐706_601_SL.TXT taccatacaa gcattagcta ttattagtag tattaaagat aaaattttca ctgagaaata 2400

caaagtaaaa ttttggactt tatcttttta ccaatagaac ttgagattta taatgctata 2460

tgacttattt tccaagatta aaagcttcat taggttgttt ttggattcag atagagcata 2520

agcataatca tccaagctcc taggctacat taggtgtgta aagctaccta gtagctgtgc 2580

cagttaagag agaatgaaca aaatctggtg ccagaaagag cttgtgccag ggtgaatcca 2640

agcccagaaa ataataggat ttaaggggac acagatgcaa tcccattgac tcaaattcta 2700

ttaattcaag acaaatctgc ttctaactac ccttctgaaa gatgtaaagg agacagctta 2760 00

cagatgttac tctagtttaa tcagagccac ataatgcaac tccagcaaca taaagatact 2820

agatgctgtt ttctgaagaa aatttctcca cattgttcat gccaaaaact taaacccgaa 2880

tttgtagaat ttgtagtggt gaattgaaag cgcaatagat ggacatatca ggggattggt 2940

attgtcttga cctacctttc ccactaaaga gtgttagaaa gatgagatta tgtgcataat 3000

ttaggggtgg tagaattcat ggaaatctaa gtttgaaacc aaaagtaatg ataaactcta 3060 00

ttcatttgtt catttaaccc tcattgcaca tttacaaaag attttagaaa ctaataaaaa 3120

tatttgattc caaggatgct atgttaatgc tataatgaga aagaaatgaa atctaattct 3180

ggctctacct acttatgtgg tcaaattctg agatttagtg tgcttattta taaagtggag 3240 00

atgatacttc actgcctact tcaaaagatg actgtgagaa gtaaatgggc ctattttgga 3300 a

gaaaattctt ttaaattgta atataccata gaaatatgaa atattatata taatatagaa 3360 e

tcaagaggcc tgtccaaaag tcctcccaaa gtattataat cttttatttc actgggacaa 3420

acatttttaa aatgcatctt aatgtagtga ttgtagaaaa gtaaaaattt aagacatatt 3480

taaaaatgtg tcttgctcaa ggctatattg agagccacta ctacatgatt attgttacct 3540 00

agtgtaaaat gttgggattg tgatagatgg catccaagag ttccttctct ctcaacattc 3600

tgtgattctt aactcttaga ctatcaaata ttataatcat agaatgtgat ttttatgcct 3660

tccacattct aatctcatct ggttctaatg attttctatg cagattggaa aagtaatcag 3720 00

cctacatctg taataggcat ttagatgcag aaagtctaac attttgcaaa gccaaattaa 3780

gctaaaacca gtgagtcaac tatcacttaa cgctagtcat aggtacttga gccctagttt 3840

ttccagtttt ataatgtaaa ctctactggt ccatctttac agtgacattg agaacagaga 3900

Page 56

50471‐706_601_SL.TXT gaatggtaaa aactacatac tgctactcca aataaaataa attggaaatt aatttctgat 3960

tctgacctct atgtaaactg agctgatgat aattattatt ctaggccaca gaactgaaac 4020

atcttcagtg tctagaagaa gaactcaaac ctctggagga agtgctaaat ttagctcaaa 4080

gcaaaaactt tcacttaaga cccagggact taatcagcaa tatcaacgta atagttctgg 4140

aactaaaggt aaggcattac tttatttgct ctcctggaaa taaaaaaaaa aaagtagggg 4200

gaaaagtacc acattttaaa gtgacataac atttttggta tttgtaaagt acccatgcat 4260

gtaattagcc tacattttaa gtacactgtg aacatgaatc atttctaatg ttaaatgatt 4320

aactggggag tataagctac tgagtttgca cctaccatct actaatggac aagcctcatc 4380

ccaaactcca tcacctttca tattaacaca aaactgggag tgagagagaa gtgactgagt 4440

tgagtttcac agaaacgcag gcaagatttt attatatatt tttcaagttc cttcacagat 4500

catttactgg aatagccaat actgagttac ctgaaaggct tttcaaatgg tgtttcctta 4560

tcatttgatg gaaggactac ccataagaga tttgtcttaa aaaaaaaaac tggagccatt 4620

aaaatggcca gtggactaaa caaacaacaa tctttttaga ggcaatccca ctttcagaat 4680

cttaagtatt tttaaatgca caggaagcat aaaatatgca agggactcag gtgatgtaaa 4740

agagattcac ttttgtcttt ttatatcccg tctcctaagg tataaaattc atgagttaat 4800

aggtatccta aataagcagc ataagtatag tagtaaaaga cattcctaaa agtaactcca 4860

gttgtgtcca aatgaatcac ttattagtgg actgtttcag ttgaattaaa aaaatacatt 4920

gagatcaatg tcatctagac attgacagat tcagttcctt atctatggca agagttttac 4980

tctaaaataa ttaacatcag aaaactcatt cttaactctt gatacaaatt taagacaaaa 5040

ccatgcaaaa atctgaaaac tgtgtttcaa aagccaaaca ctttttaaaa taaaaaaatc 5100

ccaagatatg acaatattta aacaattatg cttaagagga tacagaacac tgcaacagtt 5160

ttttaaaaga gaatacttat ttaaagggaa cactctatct cacctgcttt tgttcccagg 5220

gtaggaatca cttcaaattt gaaaagctct cttttaaatc tcactatata tcaaaatagt 5280

tgcctcctta gcttatcaac tagaggaagc gtttaaatag ctcctttcag cagagaagcc 5340

taatttctaa aaagccagtc cacagaacaa aatttctaat gtttaaagct tttaaaagtt 5400

ggcaaattca cctgcattga tactatgatg gggtagggat aggtgtaagt atttatgaag 5460

Page 57

50471‐706_601_SL.TXT 50471-706 601_SL.TXT atgttcattc acacaaattt acccaaacag gaagcatgtc ctacctagct tactctagtg 5520 atgttcatto acacaaattt acccaaacag gaagcatgto ctacctagct tactctagtg 5520

tagctcgttt cgtctttggg gaaaatataa ggagattcac ttaagtagaa aaataggaga 5580 tagctcgttt cgtctttggg gaaaatataa ggagattcac ttaagtagaa aaataggaga 5580

ctctaatcaa gatttagaaa agaagaaagt ataatgtgca tatcaattca tacatttaac 5640 ctctaatcaa gatttagaaa agaagaaagt ataatgtgca tatcaattca tacatttaac 5640

ttacacaaat ataggtgtac attcagagga aaagcgatca agtttatttc acatccagca 5700 ttacacaaat ataggtgtac attcagagga aaagcgatca agtttatttc acatccagca 5700

tttaatattt gtctagatct atttttattt aaatctttat ttgcacccaa tttagggaaa 5760 tttaatattt gtctagatct atttttattt aaatctttat ttgcacccaa tttagggaaa 5760

aaatttttgt gttcattgac tgaattaaca aatgaggaaa atctcagctt ctgtgttact 5820 aaatttttgt gttcattgac tgaattaaca aatgaggaaa atctcagctt ctgtgttact 5820

atcatttggt atcataacaa aatacgcaat tttggcattc attttgatca tttcaagaaa 5880 atcatttggt atcataacaa aatacgcaat tttggcatto attttgatca tttcaagaaa 5880

atgtgaataa ttaatatgtt tggtaagctt gaaaataaag gcaacaggcc tataagactt 5940 atgtgaataa ttaatatgtt tggtaagctt gaaaataaag gcaacaggcc tataagactt 5940

caattgggaa taactgtata taaggtaaac tactctgtac tttaaaaaat taacattttt 6000 caattgggaa taactgtata taaggtaaac tactctgtac tttaaaaaat taacattttt 6000

cttttatagg gatctgaaac aacattcatg tgtgaatatg ctgatgagac agcaaccatt 6060 cttttatagg gatctgaaac aacattcatg tgtgaatatg ctgatgagac agcaaccatt 6060

gtagaatttc tgaacagatg gattaccttt tgtcaaagca tcatctcaac actgacttga 6120 gtagaattto tgaacagatg gattaccttt tgtcaaagca tcatctcaac actgacttga 6120

taattaagtg cttcccactt aaaacatatc aggccttcta tttatttaaa tatttaaatt 6180 taattaagtg cttcccactt aaaacatatc aggccttcta tttatttaaa tatttaaatt 6180

ttatatttat tgttgaatgt atggtttgct acctattgta actattattc ttaatcttaa 6240 ttatatttat tgttgaatgt atggtttgct acctattgta actattattc ttaatcttaa 6240

aactataaat atggatcttt tatgattctt tttgtaagcc ctaggggctc taaaatggtt 6300 aactataaat atggatcttt tatgattctt tttgtaagcc ctaggggctc taaaatggtt 6300

tcacttattt atcccaaaat atttattatt atgttgaatg ttaaatatag tatctatgta 6360 tcacttattt atcccaaaat atttattatt atgttgaatg ttaaatatag tatctatgta 6360

gattggttag taaaactatt taataaattt gataaatata aacaagcctg gatatttgtt 6420 gattggttag taaaactatt taataaattt gataaatata aacaagcctg gatatttgtt 6420

attttggaaa cagcacagag taagcattta aatatttctt agttacttgt gtgaactgta 6480 attttggaaa cagcacagag taagcattta aatatttctt agttacttgt gtgaactgta 6480

ggatggttaa aatgcttaca aaagtcactc tttctctgaa gaaatatgta gaacagagat 6540 ggatggttaa aatgcttaca aaagtcacto tttctctgaa gaaatatgta gaacagagat 6540

gtagacttct caaaagccct tgctttgtcc tttcaagggc tgatcagacc cttagttctg 6600 gtagacttct caaaagccct tgctttgtcc tttcaagggc tgatcagacc cttagttctg 6600

gcatctctta gcagattata ttttccttct tcttaaaatg ccaaacacaa acactcttga 6660 gcatctctta gcagattata ttttccttct tcttaaaatg ccaaacacaa acactcttga 6660

aactcttcat agatttggtg tggc 6684 aactcttcat agatttggtg tggc 6684

<210> 96 <210> 96 <211> 1419 <211> 1419 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

Page 58 Page 58

50471‐706_601_SL.TXT

<220> <022> <221> source <IZZ> anunos <223> /note="CD19‐specific chimeric antigen receptor dso/ <EZZ> (CD19‐CD8a‐CD28‐CD3z)"

<400> 96 96 <00 gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60 09

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180 08T

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggagcag 240 DATE

gaggacatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300 00E

ggaacaaagc tggagatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360 09E

ggcagcacca agggcgaggt gaagctgcag gagagcggcc ctggcctggt ggcccccagc 420

7 cagagcctga gcgtgacctg taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 480 08/

tggatccggc agccccctag gaagggcctg gagtggctgg gcgtgatctg gggcagcgag 540 STS

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600 009

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgt 660 099

e gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720

e OZL

gtgaccgtgt ccagcaagcc caccaccacc cctgccccta gacctccaac cccagcccct 780 08L

acaatcgcca gccagcccct gagcctgagg cccgaagcct gtagacctgc cgctggcgga 840 79 gccgtgcaca ccagaggcct ggatttcgcc tgcgacatct acatctgggc ccctctggcc 900 006

ggcacctgtg gcgtgctgct gctgagcctg gtcatcaccc tgtactgcaa ccaccggaat 960 096

aggagcaagc ggagcagagg cggccacagc gactacatga acatgacccc ccggaggcct 1020 020T been ggccccaccc ggaagcacta ccagccctac gcccctccca gggacttcgc cgcctaccgg 1080 080I

agccgggtga agttcagccg gagcgccgac gcccctgcct accagcaggg ccagaaccag 1140

ctgtacaacg agctgaacct gggccggagg gaggagtacg acgtgctgga caagcggaga 1200

ggccgggacc ctgagatggg cggcaagccc cggagaaaga accctcagga gggcctgtat 1260 092T esse aacgaactgc agaaagacaa gatggccgag gcctacagcg agatcggcat gaagggcgag 1320 OZET

cggcggaggg gcaagggcca cgacggcctg taccagggcc tgagcaccgc caccaaggat 1380 08ET

Page 59 6S anded

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

acctacgacg ccctgcacat gcaggccctg ccccccaga 1419 acctacgacg ccctgcacat gcaggccctg cccccccaga 1419

<210> 97 <210> 97 <211> 1485 <211> 1485 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="CD19‐specific chimeric antigen receptor <223> /note="CD19-specific chimeric antigen receptor (CD19‐CD8a‐CD28‐CD3z) with Signal peptide" (CD19-CD8a-CD28-CD3z) with Signal peptide"

<400> 97 <400> 97 atgctgctgc tggtgaccag cctgctgctg tgtgagctgc cccaccccgc ctttctgctg 60 atgctgctgc tggtgaccag cctgctgctg tgtgagctgc cccaccccgc ctttctgctg 60

atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120 atccccgaca tccagatgac ccagaccaco tccagcctga gcgccagcct gggcgaccgg 120

gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180 gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180

aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240 aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240

cccagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300 cccagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300

gagcaggagg acatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360 gagcaggagg acatcgccac ctacttttgo cagcagggca acacactgcc ctacaccttt 360

ggcggcggaa caaagctgga gatcaccggc agcacctccg gcagcggcaa gcctggcagc 420 ggcggcggaa caaagctgga gatcaccggc agcacctccg gcagcggcaa gcctggcagc 420

ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaga gcggccctgg cctggtggcc 480 ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaga gcggccctgg cctggtggcc 480

cccagccaga gcctgagcgt gacctgtacc gtgtccggcg tgtccctgcc cgactacggc 540 cccagccaga gcctgagcgt gacctgtacc gtgtccggcg tgtccctgcc cgactacggc 540

gtgtcctgga tccggcagcc ccctaggaag ggcctggagt ggctgggcgt gatctggggc 600 gtgtcctgga tccggcagcc ccctaggaag ggcctggagt ggctgggcgt gatctggggo 600

agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660 agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660

agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720 agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720

tactgtgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780 tactgtgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780

accagcgtga ccgtgtccag caagcccacc accacccctg cccctagacc tccaacccca 840 accagcgtga ccgtgtccag caagcccacc accacccctg cccctagacc tccaacccca 840

gcccctacaa tcgccagcca gcccctgagc ctgaggcccg aagcctgtag acctgccgct 900 gcccctacaa tcgccagcca gcccctgagc ctgaggcccg aagcctgtag acctgccgct 900

ggcggagccg tgcacaccag aggcctggat ttcgcctgcg acatctacat ctgggcccct 960 ggcggagccg tgcacaccag aggcctggat ttcgcctgcg acatctacat ctgggcccct 960

Page 60 Page 60

50471‐706_601_SL.TXT 50471-706_601_SL.TXT ctggccggca cctgtggcgt gctgctgctg agcctggtca tcaccctgta ctgcaaccad ctggccggca cctgtggcgt gctgctgctg agcctggtca tcaccctgta ctgcaaccac 1020 1020

cggaatagga gcaagcggag cagaggcggc cacagcgact acatgaacat gaccccccgg cggaatagga gcaagcggag cagaggcggc cacagcgact acatgaacat gaccccccgg 1080 1080

aggcctggcc ccacccggaa gcactaccag ccctacgccc ctcccaggga cttcgccgcc aggcctggcc ccacccggaa gcactaccag ccctacgccc ctcccaggga cttcgccgcc 1140 1140

taccggagcc gggtgaagtt cagccggago gccgacgccc ctgcctacca gcagggccag taccggagcc gggtgaagtt cagccggagc gccgacgccc ctgcctacca gcagggccag 1200 1200

aaccagctgt acaacgagct gaacctgggc cggagggagg agtacgacgt gctggacaag aaccagctgt acaacgagct gaacctgggc cggagggagg agtacgacgt gctggacaag 1260 1260

cggagaggcc gggaccctga gatgggcggc aagccccgga gaaagaaccc tcaggagggo cggagaggcc gggaccctga gatgggcggc aagccccgga gaaagaaccc tcaggagggc 1320 1320

ctgtataacg aactgcagaa agacaagatg gccgaggcct acagcgagat cggcatgaag ctgtataacg aactgcagaa agacaagatg gccgaggcct acagcgagat cggcatgaag 1380 1380

ggcgagcggc ggaggggcaa gggccacgad ggcctgtacc agggcctgag caccgccaco ggcgagcggc ggaggggcaa gggccacgac ggcctgtacc agggcctgag caccgccacc 1440 1440

aaggatacct acgacgccct gcacatgcag gccctgcccc ccaga 1485 aaggatacct acgacgccct gcacatgcag gccctgcccc ccaga 1485

<210> 98 <210> 98 <211> 360 <211> 360 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti-CD19 monoclonal antibody clone FMC63 <223> /note="Anti‐CD19 monoclonal antibody clone FMC63 variable heavy chain" variable heavy chain"

<400> 98 <400> 98 gaggtgaagc tgcaggagag cggccctggc ctggtggccc ccagccagag cctgagcgtg gaggtgaagc tgcaggagag cggccctggc ctggtggccc ccagccagag cctgagcgtg 60 60

acctgtaccg tgtccggcgt gtccctgccc gactacggcg tgtcctggat ccggcagccc acctgtaccg tgtccggcgt gtccctgccc gactacggcg tgtcctggat ccggcagccc 120 120

cctaggaagg gcctggagtg gctgggcgtg atctggggca gcgagaccad ctactacaac cctaggaagg gcctggagtg gctgggcgtg atctggggca gcgagaccac ctactacaac 180 180

agcgccctga agagccggct gaccatcato aaggacaaca gcaagagcca ggtgttcctg agcgccctga agagccggct gaccatcatc aaggacaaca gcaagagcca ggtgttcctg 240 240

aagatgaaca gcctgcagad cgacgacaco gccatctact actgtgccaa gcactactad aagatgaaca gcctgcagac cgacgacacc gccatctact actgtgccaa gcactactac 300 300

tacggcggca gctacgccat ggactactgg ggccagggca ccagcgtgad cgtgtccago tacggcggca gctacgccat ggactactgg ggccagggca ccagcgtgac cgtgtccagc 360 360

<210> 99 <210> 99 <211> 735 <211> 735 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence Page 61 Page 61

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<220> <220> <221> source <221> source <223> /note="Anti‐CD19 clone FMC63 single chain fragment <223> /note="Anti-CD19 clone FMC63 single chain fragment variable (scFv) with Whitlow linker" variable (scFv) with Whitlow linker"

<400> 99 <400> 99 gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60 gacatccaga tgacccagad cacctccago ctgagcgcca gcctgggcga ccgggtgacc 60

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120 atcagctgcc gggccagcca ggacatcage aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180 gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccago 180

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggagcag 240 cggtttagcg gcagcggctc cggcaccgad tacagcctga ccatctccaa cctggagcag 240

gaggacatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300 gaggacatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300

ggaacaaagc tggagatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360 ggaacaaage tggagatcad cggcagcaco tccggcagcg gcaagcctgg cagcggcgag 360

ggcagcacca agggcgaggt gaagctgcag gagagcggcc ctggcctggt ggcccccagc 420 ggcagcacca agggcgaggt gaagctgcag gagagcggcc ctggcctggt ggcccccago 420

cagagcctga gcgtgacctg taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 480 cagagcctga gcgtgacctg taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 480

tggatccggc agccccctag gaagggcctg gagtggctgg gcgtgatctg gggcagcgag 540 tggatccggc agccccctag gaagggcctg gagtggctgg gcgtgatctg gggcagcgag 540

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600 accacctact acaacagcgc cctgaagage cggctgacca tcatcaagga caacagcaag 600

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgt 660 agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgt 660

gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720 gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccago 720

gtgaccgtgt ccagc 735 gtgaccgtgt ccago 735

<210> 100 <210> 100 <211> 333 <211> 333 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="hM195 VL" <223> /note="hM195 VL" "

Page 62 Page 62

50471‐706_601_SL.TXT 50471-706_601_SL.TXT -

<400> 100 <400> 100 gacattcaga tgacccagtc tccgagctct ctgtccgcat cagtaggaga cagggtcacc 60 gacattcaga tgacccagto tccgagctct ctgtccgcat cagtaggaga cagggtcacc 60

atcacatgca gagccagcga aagtgtcgac aattatggca ttagctttat gaactggttc 120 atcacatgca gagccagcga aagtgtcgac aattatggca ttagctttat gaactggttc 120

caacagaaac ccgggaaggc tcctaagctt ctgatttacg ctgcatccaa ccaaggctcc 180 caacagaaac ccgggaaggc tcctaagctt ctgatttacg ctgcatccaa ccaaggctcc 180

ggggtaccct ctcgcttctc aggcagtgga tctgggacag acttcactct caccatttca 240 ggggtaccct ctcgcttctc aggcagtgga tctgggacag acttcactct caccatttca 240

tctctgcagc ctgatgactt cgcaacctat tactgtcagc aaagtaagga ggttccgtgg 300 tctctgcagc ctgatgactt cgcaacctat tactgtcago aaagtaagga ggttccgtgg 300

acgttcggtc aagggaccaa ggtggagatc aaa 333 acgttcggtc aagggaccaa ggtggagatc aaa 333

<210> 101 <210> 101 <211> 348 <211> 348 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="hM195 VH" <223> /note="hM195 VH"

<400> 101 <400> 101 caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctgggagctc agtgaaggtt 60 caggttcago tggtgcagtc tggagctgag gtgaagaagc ctgggagctc agtgaaggtt 60

tcctgcaaag cttctggcta caccttcact gactacaaca tgcactgggt gaggcaggct 120 tcctgcaaag cttctggcta caccttcact gactacaaca tgcactgggt gaggcaggct 120

cctggccaag gcctggaatg gattggatat atttatcctt acaatggtgg taccggctac 180 cctggccaag gcctggaatg gattggatat atttatcctt acaatggtgg taccggctac 180

aaccagaagt tcaagagcaa ggccacaatt acagcagacg agagtactaa cacagcctac 240 aaccagaagt tcaagagcaa ggccacaatt acagcagacg agagtactaa cacagcctac 240

atggaactct ccagcctgag gtctgaggac actgcagtct attactgcgc aagagggcgc 300 atggaactct ccagcctgag gtctgaggad actgcagtct attactgcgc aagagggcgc 300

cccgctatgg actactgggg ccaagggact ctggtcactg tctcttca 348 cccgctatgg actactgggg ccaagggact ctggtcactg tctcttca 348

<210> 102 <210> 102 <211> 726 <211> 726 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

Page 63 Page 63

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<220> <220> <221> source <221> source <223> /note="hM195 scFv with linker" <223> /note="hM195 scFv with linker"

<400> 102 <400> 102 gacattcaga tgacccagtc tccgagctct ctgtccgcat cagtaggaga cagggtcacc 60 gacattcaga tgacccagto tccgagctct ctgtccgcat cagtaggaga cagggtcacc 60

caacagaaac ccgggaaggc tcctaagctt ctgatttacg ctgcatccaa ccaaggctcc 180 caacagaaac ccgggaaggc tcctaagctt ctgatttacg ctgcatccaa ccaaggctco 180

tctctgcagc ctgatgactt cgcaacctat tactgtcagc aaagtaagga ggttccgtgg 300 tctctgcago ctgatgactt cgcaacctat tactgtcagc aaagtaagga ggttccgtgg 300

acgttcggtc aagggaccaa ggtggagatc aaaggtggcg gtggctcggg cggtggtggg 360 acgttcggtc aagggaccaa ggtggagatc aaaggtggcg gtggctcggg cggtggtggg 360

tcgggtggcg gcggatctca ggttcagctg gtgcagtctg gagctgaggt gaagaagcct 420 tcgggtggcg gcggatctca ggttcagctg gtgcagtctg gagctgaggt gaagaagcct 420

gggagctcag tgaaggtttc ctgcaaagct tctggctaca ccttcactga ctacaacatg 480 gggagctcag tgaaggtttc ctgcaaagct tctggctaca ccttcactga ctacaacatg 480

cactgggtga ggcaggctcc tggccaaggc ctggaatgga ttggatatat ttatccttac 540 cactgggtga ggcaggctcc tggccaaggc ctggaatgga ttggatatat ttatccttac 540

aatggtggta ccggctacaa ccagaagttc aagagcaagg ccacaattac agcagacgag 600 aatggtggta ccggctacaa ccagaagttc aagagcaagg ccacaattac agcagacgag 600

agtactaaca cagcctacat ggaactctcc agcctgaggt ctgaggacac tgcagtctat 660 agtactaaca cagcctacat ggaactctco agcctgaggt ctgaggacac tgcagtctat 660

tactgcgcaa gagggcgccc cgctatggac tactggggcc aagggactct ggtcactgtc 720 tactgcgcaa gagggcgccc cgctatggad tactggggcc aagggactct ggtcactgto 720

tcttca 726 tcttca 726

<210> 103 <210> 103 <211> 2773 <211> 2773 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR variant III (EGFRvIII)" <223> /note="EGFR variant III (EGFRvIII)' "

<400> 103 <400> 103 atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60 atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60

gcgagtcggg ctctggagga aaagaaaggt aattatgtgg tgacagatca cggctcgtgc 120 gcgagtcggg ctctggagga aaagaaaggt aattatgtgg tgacagatca cggctcgtgc 120

Page 64 Page 64

50471‐706_601_SL.TXT gtccgagcct gtggggccga cagctatgag atggaggaag acggcgtccg caagtgtaag 180 08T

aagtgcgaag ggccttgccg caaagtgtgt aacggaatag gtattggtga atttaaagac 240

tcactctcca taaatgctac gaatattaaa cacttcaaaa actgcacctc catcagtggc 300 00E

the gatctccaca tcctgccggt ggcatttagg ggtgactcct tcacacatac tcctcctctg 360 09E

gatccacagg aactggatat tctgaaaacc gtaaaggaaa tcacagggtt tttgctgatt 420

7 caggcttggc ctgaaaacag gacggacctc catgcctttg agaacctaga aatcatacgc 480 08/

ggcaggacca agcaacatgg tcagttttct cttgcagtcg tcagcctgaa cataacatcc 540

ttgggattac gctccctcaa ggagataagt gatggagatg tgataatttc aggaaacaaa 600 009

aatttgtgct atgcaaatac aataaactgg aaaaaactgt ttgggacctc cggtcagaaa 660 099 the the accaaaatta taagcaacag aggtgaaaac agctgcaagg ccacaggcca ggtctgccat 720 02L

gccttgtgct cccccgaggg ctgctggggc ccggagccca gggactgcgt ctcttgccgg 780 08L

aatgtcagcc gaggcaggga atgcgtggac aagtgcaacc ttctggaggg tgagccaagg 840

e the gagtttgtgg agaactctga gtgcatacag tgccacccag agtgcctgcc tcaggccatg 900 006

aacatcacct gcacaggacg gggaccagac aggagtcatg ggagaaaaca acaccctggt 960 096

ctggaagtac gcagacgccg gccatgtgtg ccacctgtgc catccaaact gcacctacgg 1020 0201

atgcactggg ccaggtcttg aaggctgtcc aacgaatggg cctaagatcc cgtccatcgc 1080 080I

cactgggatg gtgggggccc tcctcttgct gctggtggtg gccctgggga tcggcctctt 1140

catgcgaagg cgccacatcg ttcggaagcg cacgctgcgg aggctgctgc aggagaggga 1200

gcttgtggag cctcttacac ccagtggaga agctcccaac caagctctct tgaggatctt 1260

See e gaaggaaact gaattcaaaa agatcaaagt gctgggctcc ggtgcgttcg gcacggtgta 1320 OZET

taagggactc tggatcccag aaggtgagaa agttaaaatt cccgtcgcta tcaaggaatt 1380 08ET

aagagaagca acatctccga aagccaacaa ggaaatcctc gatgaagcct acgtgatggc 1440

cagcgtggac aacccccacg tgtgccgcct gctgggcatc tgcctcacct ccaccgtgca 1500 00ST

gctcatcacg cagctcatgc ccttcggctg cctcctggac tatgtccggg aacacaaaga 1560 09ST

caatattggc tcccagtacc tgctcaactg gtgtgtgcag atcgcaaagg gcatgaacta 1620 The cttggaggac cgtcgcttgg tgcaccgcga cctggcagcc aggaacgtac tggtgaaaac 1680 089T

Page 65 S9 aged the

50471‐706_601_SL.TXT 50471-706_601_SL.TXT accgcagcat gtcaagatca cagattttgg gctggccaaa ctgctgggtg cggaagagaa 1740 accgcagcat gtcaagatca cagattttgg gctggccaaa ctgctgggtg cggaagagaa 1740

agaataccat gcagaaggag gcaaagtgcc tatcaagtgg atggcattgg aatcaatttt 1800 agaataccat gcagaaggag gcaaagtgcc tatcaagtgg atggcattgg aatcaatttt 1800

acacagaatc tatacccacc agagtgatgt ctggagctac ggggtgactg tttgggagtt 1860 acacagaatc tatacccacc agagtgatgt ctggagctac ggggtgactg tttgggagtt 1860

gatgaccttt ggatccaagc catatgacgg aatccctgcc agcgagatct cctccatcct 1920 gatgaccttt ggatccaagc catatgacgg aatccctgcc agcgagatct cctccatcct 1920

ggagaaagga gaacgcctcc ctcagccacc catatgtacc atcgatgtct acatgatcat 1980 ggagaaagga gaacgcctcc ctcagccacc catatgtacc atcgatgtct acatgatcat 1980

ggtcaagtgc tggatgatag acgcagatag tcgcccaaag ttccgtgagt tgatcatcga 2040 ggtcaagtgc tggatgatag acgcagatag tcgcccaaag ttccgtgagt tgatcatcga 2040

attctccaaa atggcccgag acccccagcg ctaccttgtc attcaggggg atgaaagaat 2100 attctccaaa atggcccgag acccccagcg ctaccttgtc attcaggggg atgaaagaat 2100

gcatttgcca agtcctacag actccaactt ctaccgtgcc ctgatggatg aagaagacat 2160 gcatttgcca agtcctacag actccaactt ctaccgtgcc ctgatggatg aagaagacat 2160

ggacgacgtg gtggatgccg acgagtacct catcccacag cagggcttct tcagcagccc 2220 ggacgacgtg gtggatgccg acgagtacct catcccacag cagggcttct tcagcagccc 2220

ctccacgtca cggactcccc tcctgagctc tctgagtgca accagcaaca attccaccgt 2280 ctccacgtca cggactcccc tcctgagctc tctgagtgca accagcaaca attccaccgt 2280

ggcttgcatt gatagaaatg ggctgcaaag ctgtcccatc aaggaagaca gcttcttgca 2340 ggcttgcatt gatagaaatg ggctgcaaag ctgtcccatc aaggaagaca gcttcttgca 2340

gcgatacagc tcagacccca caggcgcctt gactgaggac agcatagacg acaccttcct 2400 gcgatacagc tcagacccca caggcgcctt gactgaggac agcatagacg acaccttcct 2400

cccagtgcct gaatacataa accagtccgt tcccaaaagg cccgctggct ctgtgcagaa 2460 cccagtgcct gaatacataa accagtccgt tcccaaaaagg cccgctggct ctgtgcagaa 2460

tcctgtctat cacaatcagc ctctgaaccc cgcgcccagc agagacccac actaccagga 2520 tcctgtctat cacaatcagc ctctgaaccc cgcgcccago agagacccao actaccagga 2520

cccccacagc actgcagtgg gcaaccccga gtatctcaac actgtccagc ccacctgtgt 2580 cccccacaga actgcagtgg gcaaccccga gtatctcaac actgtccagc ccacctgtgt 2580

caacagcaca ttcgacagcc ctgcccactg ggcccagaaa ggcagccacc aaattagcct 2640 caacagcaca ttcgacagcc ctgcccactg ggcccagaaa ggcagccacc aaattagcct 2640

ggacaaccct gactaccagc aggacttctt tcccaaggaa gccaagccaa atggcatctt 2700 ggacaaccct gactaccago aggacttctt tcccaaggaa gccaagccaa atggcatctt 2700

taagggctcc acagctgaaa atgcagaata cctaagggtc gcgccacaaa gcagtgaatt 2760 taagggctcc acagctgaaa atgcagaata cctaagggtc gcgccacaaa gcagtgaatt 2760

tattggagca tga 2773 tattggagca tga 2773

<210> 104 <210> 104 <211> 348 <211> 348 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note=' Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone 139 VH" <223> /note="Anti- EGFRVIII Clone 139 VH" Page 66 Page 66

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<400> 104 <400> 104 gaagtgcagg tgctggaaag cggcggagga ctggtgcagc ctggcggatc tctgagactg 60 gaagtgcagg tgctggaaag cggcggagga ctggtgcagc ctggcggatc tctgagactg 60

agctgtgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120 agctgtgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120

cctggaaaag gcctggaatg ggtgtccgcc atctctggct ccggcggcag caccaattac 180 cctggaaaag gcctggaatg ggtgtccgcc atctctggct ccggcggcag caccaattac 180

gccgatagcg tgaagggccg gttcaccatc agccgggaca acagcaagaa caccctgtac 240 gccgatagcg tgaagggccg gttcaccato agccgggaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgag agccgaggac accgccgtgt actactgtgc cggaagctct 300 ctgcagatga acagcctgag agccgaggad accgccgtgt actactgtgc cggaagctct 300

gggtggagcg agtattgggg ccagggcaca ctcgtgaccg tgtccagc 348 gggtggagcg agtattggggg ccagggcaca ctcgtgaccg tgtccagc 348

<210> 105 <210> 105 <211> 321 <211> 321 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone 139 VL" <223> /note="Anti- EGFRVIII Clone 139 VL"

<400> 105 <400> 105 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga cagagtgacc 60

atcacctgta gagccagcca gggcatccgg aacaacctgg cctggtatca gcagaagccc 120 atcacctgta gagccagcca gggcatccgg aacaacctgg cctggtatca gcagaagccc 120

ggcaaggccc ccaagcggct gatctacgcc gccagcaatc tgcagagcgg cgtgccctct 180 ggcaaggccc ccaagcggct gatctacgcc gccagcaatc tgcagagcgg cgtgccctct 180

agattcaccg gctctggcag cggcaccgag ttcaccctga tcgtgtctag cctgcagccc 240 agattcaccg gctctggcag cggcaccgag ttcaccctga tcgtgtctag cctgcagccc 240

gaggacttcg ccacctacta ctgcctgcag caccacagct accccctgac atctggcgga 300 gaggacttcg ccacctacta ctgcctgcag caccacagct accccctgac atctggcgga 300

ggcaccaagg tggaaatcaa g 321 ggcaccaagg tggaaatcaa g 321

<210> 106 <210> 106 <211> 360 <211> 360 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

Page 67 Page 67

50471‐706_601_SL.TXT 50471-706_601_SL.TXT -

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone MR1 VH" <223> /note="Anti- EGFRVIII Clone MR1 VH"

<400> 106 <400> 106 caagtgaagc tgcagcagtc tggcggaggc ctcgtgaaac ctggcgcctc tctgaagctg 60 caagtgaago tgcagcagto tggcggaggo ctcgtgaaac ctggcgcctc tctgaagctg 60

agctgcgtga ccagcggctt caccttcaga aagttcggca tgagctgggt gcgccagacc 120 agctgcgtga ccagcggctt caccttcaga aagttcggca tgagctgggt gcgccagaco 120

agcgacaagc ggctggaatg ggtggccagc atcagcaccg gcggctacaa cacctactac 180 agcgacaagc ggctggaatg ggtggccago atcagcaccg gcggctacaa cacctactac 180

agcgacaacg tgaagggcag attcaccatc agcagagaga acgccaagaa taccctgtac 240 agcgacaacg tgaagggcag attcaccato agcagagaga acgccaagaa taccctgtad 240

ctgcagatga gcagcctgaa gtccgaggac accgccctgt actactgcac cagaggctac 300 ctgcagatga gcagcctgaa gtccgaggad accgccctgt actactgcad cagaggctac 300

agcagcacca gctacgccat ggactattgg ggccagggca ccaccgtgac cgtgtctagt 360 agcagcacca gctacgccat ggactattgg ggccagggca ccaccgtgac cgtgtctagt 360

<210> 107 <210> 107 <211> 321 <211> 321 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone MR1 VL" <223> /note="Anti- EGFRVIII Clone MR1 VL" "

<400> 107 <400> 107 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60

atccggtgca tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 atccggtgca tgaccagcac cgacatcgad gacgacatga actggtatca gcagaagccc 120

ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaagc 180 ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaagc 180

agattcagca gctctggcac cggcaccgac ttcgtgttca ccatcgagaa caccctgagc 240 agattcagca gctctggcac cggcaccgac ttcgtgttca ccatcgagaa caccctgago 240

gaggacgtgg gcgactacta ctgcctgcag agcttcaacg tgcccctgac ctttggcgac 300 gaggacgtgg gcgactacta ctgcctgcag agcttcaacg tgcccctgac ctttggcgac 300

ggcaccaagc tggaaatcaa g 321 ggcaccaago tggaaatcaa g 321

<210> 108 <210> 108 <211> 360 <211> 360 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

Page 68 Page 68

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone MR1‐1 VH" <223> /note="Anti- EGFRVIII Clone MR1-1 VH"

<400> 108 <400> 108 caagtgaagc tgcagcagtc tggcggaggc ctcgtgaaac ctggcgcctc tctgaagctg 60 caagtgaagc tgcagcagto tggcggaggo ctcgtgaaac ctggcgcctc tctgaagctg 60

agctgcgtga ccagcggctt caccttcaga aagttcggca tgagctgggt gcgccagacc 120 agctgcgtga ccagcggctt caccttcaga aagttcggca tgagctgggt gcgccagacc 120

agcgacaagc ggctggaatg ggtggccagc atcagcaccg gcggctacaa cacctactac 180 agcgacaago ggctggaatg ggtggccagc atcagcaccg gcggctacaa cacctactac 180

agcgacaacg tgaagggcag attcaccatc agcagagaga acgccaagaa taccctgtac 240 agcgacaacg tgaagggcag attcaccato agcagagaga acgccaagaa taccctgtac 240

agcccctaca gctacgccat ggactattgg ggccagggca ccaccgtgac cgtgtctagt 360 agcccctaca gctacgccat ggactattgg ggccagggca ccaccgtgac cgtgtctagt 360

<210> 109 <210> 109 <211> 321 <211> 321 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone MR1‐1 VL" <223> /note="Anti- EGFRVIII Clone MR1-1 VL"

<400> 109 <400> 109 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgaco 60

atccggtgca tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 atccggtgca tgaccagcad cgacatcgad gacgacatga actggtatca gcagaagccc 120

ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaagc 180 ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaago 180

gaggacgtgg gcgactacta ctgcctgcag agctggaacg tgcccctgac ctttggcgac 300 gaggacgtgg gcgactacta ctgcctgcag agctggaacg tgcccctgac ctttggcgac 300

ggcaccaagc tggaaatcaa g 321 ggcaccaage tggaaatcaa g 321

<210> 110 <210> 110 Page 69 Page 69

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <211> 360 <211> 360 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone humMR1‐1 VH" <223> /note="Anti- EGFRVIII Clone humMR1-1 VH"

<400> 110 <400> 110 caggtgcagc tgcaggaatc tggcggaggg ctcgtgaagc ctggcggaag cctgaagctg 60 caggtgcagc tgcaggaatc tggcggaggg ctcgtgaagc ctggcggaag cctgaagctg 60

agctgtgccg ccagcggctt caccttcagc aagttcggca tgagctgggt gcgccagacc 120 agctgtgccg ccagcggctt caccttcagc aagttcggca tgagctgggt gcgccagacc 120

cccgacaaga gactggaatg ggtggccagc atcagcaccg gcggctacaa tacctactac 180 cccgacaaga gactggaatg ggtggccago atcagcaccg gcggctacaa tacctactac 180

agcgacaacg tgaagggccg gttcaccatc tcccgggaca acgccaagaa caccctgtac 240 agcgacaacg tgaagggccg gttcaccatc tcccgggaca acgccaagaa caccctgtad 240

ctgcagatga gcagcctgaa gtccgaggac accgccatgt actactgtgc cagaggctac 300 ctgcagatga gcagcctgaa gtccgaggad accgccatgt actactgtgc cagaggctac 300

agcccctaca gctacgccat ggattactgg ggccagggca caatggtcac cgtgtcctct 360 agcccctaca gctacgccat ggattactgg ggccagggca caatggtcac cgtgtcctct 360

<210> 111 <210> 111 <211> 321 <211> 321 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone humMR1‐1 VL" <223> /note="Anti- EGFRVIII Clone humMR1-1 VL"

<400> 111 <400> 111 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga cagagtgacc 60

atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 atcacctgta tgaccagcad cgacatcgad gacgacatga actggtatca gcagaagccc 120

ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccagc 180 ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccagc 180

agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240 agattttctg gcagcggctc cggcaccgad ttcatcttca ccatcagctc cctgcagccc 240

gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga 300 gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga 300

Page 70 Page 70

50471‐706_601_SL.TXT 50471-706_601_SL.TXT ggcaccaagg tggaaatcaa g 321 ggcaccaagg tggaaatcaa g 321

<210> 112 <210> 112 <211> 360 <211> 360 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone humMR1‐2 VH" <223> /note="Anti- EGFRVIII Clone humMR1-2 VH"

<400> 112 <400> 112 caggtgcagc tgcaggaatc tggcggaggg ctcgtgaagc ctggcggaag cctgaagctg 60 caggtgcago tgcaggaatc tggcggaggg ctcgtgaagc ctggcggaag cctgaagctg 60

agctgtgccg ccagcggctt caccttcagc aagttcggca tgagctgggt gcgccagacc 120 agctgtgccg ccagcggctt caccttcago aagttcggca tgagctgggt gcgccagacc 120

cccgacaaga gactggaatg ggtggccagc atcagcaccg gcggctacaa caccttctac 180 cccgacaaga gactggaatg ggtggccago atcagcaccg gcggctacaa caccttctac 180

agcccctaca gcttcgccat ggattactgg ggccagggca caatggtcac cgtgtcctct 360 agcccctaca gcttcgccat ggattactgg ggccagggca caatggtcac cgtgtcctct 360

<210> 113 <210> 113 <211> 321 <211> 321 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone humMR1‐2 VL" <223> /note="Anti- EGFRVIII Clone humMR1-2 VL"

<400> 113 <400> 113 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga cagagtgacc 60

Page 71 Page 71

50471‐706_601_SL.TXT 50471-706_601_SL.TXT agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240 agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240

ggcaccaagg tggaaatcaa g 321 ggcaccaagg tggaaatcaa g 321

<210> 114 <210> 114 <211> 723 <211> 723 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII scFv Clone 139" <223> /note="Anti- EGFRVIII scFv Clone 139"

<400> 114 <400> 114 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga cagagtgaco 60

ggcaaggccc ccaagcggct gatctacgcc gccagcaatc tgcagagcgg cgtgccctct 180 ggcaaggccc ccaagcggct gatctacgco gccagcaato tgcagagcgg cgtgccctct 180

gaggacttcg ccacctacta ctgcctgcag caccacagct accccctgac atctggcgga 300 gaggacttcg ccacctacta ctgcctgcag caccacagct accccctgad atctggcgga 300

ggcaccaagg tggaaatcaa gggcagcaca agcggcagcg gaaaacctgg atctggcgag 360 ggcaccaagg tggaaatcaa gggcagcaca agcggcagcg gaaaacctgg atctggcgag 360

ggctctacca agggcgaagt gcaggtgctg gaaagcggcg gaggactggt gcagcctggc 420 ggctctacca agggcgaagt gcaggtgctg gaaagcggcg gaggactggt gcagcctggc 420

ggatctctga gactgagctg tgccgccagc ggcttcacct tcagcagcta cgccatgagc 480 ggatctctga gactgagctg tgccgccagc ggcttcacct tcagcagcta cgccatgagc 480

tgggtgcgcc aggcccctgg aaaaggcctg gaatgggtgt ccgccatctc tggctccggc 540 tgggtgcgcc aggcccctgg aaaaggcctg gaatgggtgt ccgccatctc tggctccggc 540

ggcagcacca attacgccga tagcgtgaag ggccggttca ccatcagccg ggacaacagc 600 ggcagcacca attacgccga tagcgtgaag ggccggttca ccatcagccg ggacaacago 600

aagaacaccc tgtacctgca gatgaacagc ctgagagccg aggacaccgc cgtgtactac 660 aagaacaccc tgtacctgca gatgaacagc ctgagagccg aggacaccgo cgtgtactac 660

tgtgccggaa gctctgggtg gagcgagtat tggggccagg gcacactcgt gaccgtgtcc 720 tgtgccggaa gctctgggtg gagcgagtat tggggccagg gcacactcgt gaccgtgtcc 720

agc 723 agc 723

<210> 115 <210> 115 <211> 726 <211> 726 <212> DNA <212> DNA Page 72 Page 72

<220> <220> <221> source <221> source <223> /note="Anti‐EGFRvIII scFv clone MR1" <223> /note="Anti-EGFRvIII scFv clone MR1"

<400> 115 <400> 115 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 gacatcgagc tgacacagag ccctgccago ctgtctgtgg ccaccggcga gaaagtgacc 60

ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaagc 180 ggcgagcccc ccaagttcct gatcagcgag ggcaacacao tgcggcctgg cgtgccaagc 180

agattcagca gctctggcac cggcaccgac ttcgtgttca ccatcgagaa caccctgagc 240 agattcagca gctctggcac cggcaccgad ttcgtgttca ccatcgagaa caccctgagc 240

ggcaccaagc tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 ggcaccaage tggaaatcaa gggcggaggc ggatctggcg gcggaggato tgggggaggo 360

ggctctcaag tgaagctgca gcagtctggc ggaggcctcg tgaaacctgg cgcctctctg 420 ggctctcaag tgaagctgca gcagtctggc ggaggcctcg tgaaacctgg cgcctctctg 420

aagctgagct gcgtgaccag cggcttcacc ttcagaaagt tcggcatgag ctgggtgcgc 480 aagctgagct gcgtgaccag cggcttcacc ttcagaaagt tcggcatgag ctgggtgcgc 480

cagaccagcg acaagcggct ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540 cagaccagcg acaagcggct ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540

tactacagcg acaacgtgaa gggcagattc accatcagca gagagaacgc caagaatacc 600 tactacagcg acaacgtgaa gggcagatto accatcagca gagagaacgc caagaatacc 600

ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccctgtacta ctgcaccaga 660 ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccctgtacta ctgcaccaga 660

ggctacagca gcaccagcta cgccatggac tattggggcc agggcaccac cgtgaccgtg 720 ggctacagca gcaccagcta cgccatggad tattggggcc agggcaccac cgtgaccgtg 720

tctagt 726 tctagt 726

<210> 116 <210> 116 <211> 726 <211> 726 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti EGFRvIII scFv clone MR1‐1" <223> /note="Anti EGFRVIII scFv clone MR1-1" Page 73 Page 73

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<400> 116 <400> 116 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60

gaggacgtgg gcgactacta ctgcctgcag agctggaacg tgcccctgac ctttggcgac 300 gaggacgtgg gcgactacta ctgcctgcag agctggaacg tgcccctgac ctttggcgad 300

cagaccagcg acaagcggct ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540 cagaccagcg acaagcggct ggaatgggtg gccagcatca gcaccggcgg ctacaacaco 540

ggctacagcc cctacagcta cgccatggac tattggggcc agggcaccac cgtgaccgtg 720 ggctacagcc cctacagcta cgccatggad tattggggcc agggcaccac cgtgaccgtg 720

tctagt 726 tctagt 726

<210> 117 <210> 117 <211> 726 <211> 726 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐EGFRvIII scFv clone huMR1‐1" <223> /note="Anti-EGFRvIII scFv clone huMR1-1"

<400> 117 <400> 117 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcago ctgtctgcca gcgtgggcga cagagtgacc 60

ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccagc 180 ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccago 180

Page 74 Page 74

50471‐706_601_SL.TXT 50471-706_601_SL.TXT gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga 300 gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga 300

ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 ggcaccaagg tggaaatcaa gggcggaggo ggatctggcg gcggaggatc tgggggaggc 360

ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg cggaagcctg 420 ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg cggaagcctg 420

aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag ctgggtgcgc 480 aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag ctgggtgcgc 480

cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaatacc 540 cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaatacc 540

tactacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacacc 600 tactacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacacc 600

ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta ctgtgccaga 660 ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta ctgtgccaga 660

ggctacagcc cctacagcta cgccatggat tactggggcc agggcacaat ggtcaccgtg 720 ggctacagcc cctacagcta cgccatggat tactggggcc agggcacaat ggtcaccgtg 720

tcctct 726 tcctct 726

<210> 118 <210> 118 <211> 726 <211> 726 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐EGFRvIII scFv clone huMR1‐2" <223> /note="Anti-EGFRvIII scFv clone huMR1-2"

<400> 118 <400> 118 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga cagagtgacc 60

atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 atcacctgta tgaccagcac cgacatogac gacgacatga actggtatca gcagaagccc 120

agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240 agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240

ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360

cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540 cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540

Page 75 Page 75

50471‐706_601_SL.TXT ttctacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacacc 600

ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta ctgtgccaga 660

ggctacagcc cctacagctt cgccatggat tactggggcc agggcacaat ggtcaccgtg 720

tcctct 726

<210> 119 <211> 1410 <212> DNA <213> Artificial Sequence

<220> <221> source <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

<220> <221> source <223> /note="EGFRvIII CAR (clone 139 scFv.CD8alpha hinge &TM.4‐1BB.CD3‐zeta)"

<400> 119 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60

atcacctgta gagccagcca gggcatccgg aacaacctgg cctggtatca gcagaagccc 120

ggcaaggccc ccaagcggct gatctacgcc gccagcaatc tgcagagcgg cgtgccctct 180

agattcaccg gctctggcag cggcaccgag ttcaccctga tcgtgtctag cctgcagccc 240

gaggacttcg ccacctacta ctgcctgcag caccacagct accccctgac atctggcgga 300

ggcaccaagg tggaaatcaa gggcagcaca agcggcagcg gaaaacctgg atctggcgag 360

ggctctacca agggcgaagt gcaggtgctg gaaagcggcg gaggactggt gcagcctggc 420

ggatctctga gactgagctg tgccgccagc ggcttcacct tcagcagcta cgccatgagc 480

tgggtgcgcc aggcccctgg aaaaggcctg gaatgggtgt ccgccatctc tggctccggc 540

ggcagcacca attacgccga tagcgtgaag ggccggttca ccatcagccg ggacaacagc 600

aagaacaccc tgtacctgca gatgaacagc ctgagagccg aggacaccgc cgtgtactac 660

tgtgccggaa gctctgggtg gagcgagtat tggggccagg gcacactcgt gaccgtgtcc 720

agcaagccca ccaccacccc tgcccctaga cctccaaccc cagcccctac aatcgccagc 780

cagcccctga gcctgaggcc cgaagcctgt agacctgccg ctggcggagc cgtgcacacc 840 Page 76

50471-786_601_SL.TXT

50471‐706_601_SL.TXT

agaggcctgg atttcgcctg cgacatctac atctgggccc ctctggccgg cacctgtggc 900 900

gtgctgctgc tgagcctggt catcaccctg tactgcaacc accggaataa gagaggccgg 960 960

aagaaactgc tgtacatctt caagcagccc ttcatgcggc ccgtgcagac cacccaggaa 1020 1020

gaggacggct gcagctgccg gttccccgag gaagaggaag gcggctgcga actgcgggtg 1080 1080

aagttcagcc ggagcgccga cgcccctgcc taccagcagg gccagaacca gctgtacaac 1140 1140

gagctgaacc tgggccggag ggaggagtac gacgtgctgg acaagcggag aggccgggac 1200 1200

cctgagatgg gcggcaagcc ccggagaaag aaccctcagg agggcctgta taacgaactg 1260 1260

cagaaagaca agatggccga ggcctacagc gagatcggca tgaagggcga gcggcggagg 1320 1320

ggcaagggcc acgacggcct gtaccagggc ctgagcaccg ccaccaagga tacctacgac 1380 1380 gccctgcaca gccctgcaca tgcaggccct gccccccaga 1410 1410

<210> 120 <210> <211> 1413 <211> 1413 <212> DNA Artificial <212> DNA <213> Artificial Sequence <213>

<220> <220> <221> source <221> <223> /note="Description of Artificial Sequence: Synthetic <223> polynucleotide"

<220> <220> <221> <221> source <223> /note="EGFRvIII CAR (MR1 scFv.CD8alpha hinge &TM.4‐1BB.CD3‐zeta)"

<400> 120 <400> gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 60

atccggtgca tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 120

ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaagc 180 180

agattcagca gctctggcac cggcaccgac ttcgtgttca ccatcgagaa caccctgagc 240 240

gaggacgtgg gcgactacta ctgcctgcag agcttcaacg tgcccctgac ctttggcgac 300 300

ggcaccaagc tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 360

ggctctcaag tgaagctgca gcagtctggc ggaggcctcg tgaaacctgg cgcctctctg 420 420

Page 77 Page 77

50471‐706_601_SL.TXT 50471-706_601_SL.TXT aagctgagct gcgtgaccag cggcttcacc ttcagaaagt tcggcatgag ctgggtgcgc 480 aagctgagct gcgtgaccag cggcttcacc ttcagaaagt tcggcatgag ctgggtgcgc 480

tactacagcg acaacgtgaa gggcagattc accatcagca gagagaacgc caagaatacc 600 tactacagcg acaacgtgaa gggcagattc accatcagca gagagaacgc caagaatacc 600

ggctacagca gcaccagcta cgccatggac tattggggcc agggcaccac cgtgaccgtg 720 ggctacagca gcaccagcta cgccatggac tattggggcc agggcaccac cgtgaccgtg 720

tctagtaagc ccaccaccac ccctgcccct agacctccaa ccccagcccc tacaatcgcc 780 tctagtaagc ccaccaccac ccctgcccct agacctccaa ccccagcccc tacaatcgcc 780

agccagcccc tgagcctgag gcccgaagcc tgtagacctg ccgctggcgg agccgtgcac 840 agccagcccc tgagcctgag gcccgaagcc tgtagacctg ccgctggcgg agccgtgcac 840

accagaggcc tggatttcgc ctgcgacatc tacatctggg cccctctggc cggcacctgt 900 accagaggcc tggatttcgc ctgcgacatc tacatctggg cccctctggc cggcacctgt 900

ggcgtgctgc tgctgagcct ggtcatcacc ctgtactgca accaccggaa taagagaggc 960 ggcgtgctgc tgctgagcct ggtcatcacc ctgtactgca accaccggaa taagagaggc 960

cggaagaaac tgctgtacat cttcaagcag cccttcatgc ggcccgtgca gaccacccag 1020 cggaagaaac tgctgtacat cttcaagcag cccttcatgc ggcccgtgca gaccacccag 1020

gaagaggacg gctgcagctg ccggttcccc gaggaagagg aaggcggctg cgaactgcgg 1080 gaagaggacg gctgcagctg ccggttcccc gaggaagagg aaggcggctg cgaactgcgg 1080

gtgaagttca gccggagcgc cgacgcccct gcctaccagc agggccagaa ccagctgtac 1140 gtgaagttca gccggagcgc cgacgcccct gcctaccagc agggccagaa ccagctgtac 1140

aacgagctga acctgggccg gagggaggag tacgacgtgc tggacaagcg gagaggccgg 1200 aacgagctga acctgggccg gagggaggag tacgacgtgc tggacaagcg gagaggccgg 1200

gaccctgaga tgggcggcaa gccccggaga aagaaccctc aggagggcct gtataacgaa 1260 gaccctgaga tgggcggcaa gccccggaga aagaaccctc aggagggcct gtataacgaa 1260

ctgcagaaag acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcggcgg 1320 ctgcagaaag acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcggcgg 1320

aggggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggatacctac 1380 aggggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggatacctac 1380

gacgccctgc acatgcaggc cctgcccccc aga 1413 gacgccctgc acatgcaggc cctgcccccc aga 1413

<210> 121 <210> 121 <211> 1413 <211> 1413 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (MR1‐1 scFv.CD8alpha hinge <223> /note="EGFRvIII CAR (MR1-1 scFv. CD8alpha hinge &TM.4‐1BB.CD3‐zeta)" &TM.4-1BB.CD3-zeta)" "

<400> 121 <400> 121 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 gacatogagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 Page 78 Page 78

50471‐706_601_SL.TXT

atccggtgca tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120

ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaagc 180 08T

agattcagca gctctggcac cggcaccgac ttcgtgttca ccatcgagaa caccctgagc 240

gaggacgtgg gcgactacta ctgcctgcag agctggaacg tgcccctgac ctttggcgac 300 00E

ggcaccaagc tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 088e88,888 09E

ggctctcaag tgaagctgca gcagtctggc ggaggcctcg tgaaacctgg cgcctctctg 420

aagctgagct gcgtgaccag cggcttcacc ttcagaaagt tcggcatgag ctgggtgcgc 480 08/

cagaccagcg acaagcggct ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540

tactacagcg acaacgtgaa gggcagattc accatcagca gagagaacgc caagaatacc 600 009

ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccctgtacta ctgcaccaga 660 099

ggctacagcc cctacagcta cgccatggac tattggggcc agggcaccac cgtgaccgtg 720 OZL

tctagtaagc ccaccaccac ccctgcccct agacctccaa ccccagcccc tacaatcgcc 780 08L

agccagcccc tgagcctgag gcccgaagcc tgtagacctg ccgctggcgg agccgtgcac 840 79 accagaggcc tggatttcgc ctgcgacatc tacatctggg cccctctggc cggcacctgt 900 006

ggcgtgctgc tgctgagcct ggtcatcacc ctgtactgca accaccggaa taagagaggc 960 0879878588 096

cggaagaaac tgctgtacat cttcaagcag cccttcatgc ggcccgtgca gaccacccag 1020 020T

gaagaggacg gctgcagctg ccggttcccc gaggaagagg aaggcggctg cgaactgcgg 1080 88edee99e8 080T

gtgaagttca gccggagcgc cgacgcccct gcctaccagc agggccagaa ccagctgtac 1140

aacgagctga acctgggccg gagggaggag tacgacgtgc tggacaagcg gagaggccgg 1200 002I

gaccctgaga tgggcggcaa gccccggaga aagaaccctc aggagggcct gtataacgaa 1260 092T

9997 ctgcagaaag acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcggcgg 1320 OZET

aggggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggatacctac 1380 08ET

gacgccctgc acatgcaggc cctgcccccc aga 1413 ege ETD

<210> 122 ZZI <0TZ> <211> 1413 ETD <III> <212> DNA ANC <ZIZ> <213> Artificial Sequence <ETZ> Page 79 6L aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<220> <220> <221> source <221> /note= <223> source "Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (humMR1-1 scFv. CD8alpha hinge <223> /note="EGFRvIII CAR (humMR1‐1 scFv.CD8alpha hinge &TM.4‐1BB.CD3‐zeta)" &TM.4-1BB.CD3-zeta) "

<400> 122 <400> 122 gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga cagagtgacc gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 60

atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 120 ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccagc ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccagc 180 180 agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240 240 gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga 300 300

ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 360 ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg cggaagcctg ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg cggaagcctg 420 420 aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag ctgggtgcgc aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag ctgggtgcgc 480 480 cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaatacc cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaatacc 540 540

tactacagcg acaacgtgaa gggccggttc accatctccc gggacaacgo caagaacacc tactacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacacc 600 600 ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta ctgtgccaga ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta ctgtgccaga 660 660 ggctacagcc cctacagcta cgccatggat tactggggcc agggcacaat ggtcaccgtg ggctacagcc cctacagcta cgccatggat tactggggcc agggcacaat ggtcaccgtg 720 720 tcctctaagc ccaccaccac ccctgcccct agacctccaa ccccagcccc tacaatcgcc tcctctaagc ccaccaccac ccctgcccct agacctccaa ccccagcccc tacaatcgcc 780 780 agccagcccc tgagcctgag gcccgaagcc tgtagacctg ccgctggcgg agccgtgcac agccagcccc tgagcctgag gcccgaagcc tgtagacctg ccgctggcgg agccgtgcac 840 840 accagaggcc tggatttcgc ctgcgacatc tacatctggg ccccctctggc cggcacctgt accagaggcc tggatttcgc ctgcgacatc tacatctggg cccctctggc cggcacctgt 900 900 ggcgtgctgc tgctgagcct ggtcatcacc ctgtactgca accaccggaa taagagaggo ggcgtgctgc tgctgagcct ggtcatcacc ctgtactgca accaccggaa taagagaggc 960 960

cggaagaaac tgctgtacat cttcaagcag cccttcatgc ggcccgtgca gaccacccag cggaagaaac tgctgtacat cttcaagcag cccttcatgc ggcccgtgca gaccacccag 1020 1020 gaagaggacg gctgcagctg ccggttcccc gaggaagagg aaggcggctg cgaactgcgg gaagaggacg gctgcagctg ccggttcccc gaggaagagg aaggcggctg cgaactgcgg 1080 1080 gtgaagttca gccggagcgc cgacgcccct gcctaccagc agggccagaa ccagctgtac gtgaagttca gccggagcgc cgacgcccct gcctaccagc agggccagaa ccagctgtac 1140 1140

aacgagctga acctgggccg gagggaggag tacgacgtgc tggacaagcg gagaggccgg aacgagctga acctgggccg gagggaggag tacgacgtgc tggacaagcg gagaggccgg 1200 1200

Page 80 Page 80

50471‐706_601_SL.TXT 1X1 7S T09) gaccctgaga tgggcggcaa gccccggaga aagaaccctc aggagggcct gtataacgaa 1260 checked ctgcagaaag acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcggcgg 1320 OZET

aggggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggatacctac 1380 08ET

gacgccctgc acatgcaggc cctgcccccc aga 1413 ege

<210> 123 EZI <0IZ> <211> 1413 <III> <212> DNA ANC <213> Artificial Sequence <ETZ>

<220> <022> <221> source <<<z> <223> /note="Description of Artificial Sequence: Synthetic to the <EZZ> polynucleotide"

<220> <022> <221> source <IZZ> <223> /note="EGFRvIII CAR (humMR1‐2 scFv.CD8alpha hinge Sauty e <EZZ> &TM.4‐1BB.CD3‐zeta)"

<400> 123 EZI <00 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 09

atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 OZI

checked the ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccagc 180 08T

agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240

the gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga 300 00E

ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 09E

ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg cggaagcctg 420

7 aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag ctgggtgcgc 480 08/7

cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540

ttctacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacacc 600 009

ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta ctgtgccaga 660 099

ggctacagcc cctacagctt cgccatggat tactggggcc agggcacaat ggtcaccgtg 720 02L

tcctctaagc ccaccaccac ccctgcccct agacctccaa ccccagcccc tacaatcgcc 780 08L

agccagcccc tgagcctgag gcccgaagcc tgtagacctg ccgctggcgg agccgtgcac 840 Page 81 T8 aged

58471-706_601_5L.TXT

50471‐706_601_SL.TXT

accagaggcc tggatttcgc ctgcgacatc tacatctggg cccctctggc cggcacctgt 900 900

ggcgtgctgc tgctgagcct ggtcatcacc ctgtactgca accaccggaa taagagaggc 960 960

cggaagaaac tgctgtacat cttcaagcag cccttcatgc ggcccgtgca gaccacccag 1020

gaagaggacg gctgcagctg ccggttcccc gaggaagagg aaggcggctg cgaactgcgg 1080 1080

1140 gtgaagttca gccggagcgc cgacgcccct gcctaccagc agggccagaa ccagctgtac 1140

aacgagctga acctgggccg gagggaggag tacgacgtgc tggacaagcg gagaggccgg 1200 1200

gaccctgaga tgggcggcaa gccccggaga aagaaccctc aggagggcct gtataacgaa 1260 1260

ctgcagaaag acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcggcgg 1320 1320

aggggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggatacctac 1380 1380

gacgccctgc acatgcaggc cctgcccccc aga 1413 1413

<210> <210> 124 <211> 1554 <211> 1554 <212> <212> DNA DNA <213> <213> Artificial Sequence

<220> <220> <221> <221> source <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

<220> <220> <221> source <223> /note="EGFRvIII CAR (MR1‐1 scFv.CD8alpha 2x hinge &TM.4‐1BB.CD3‐zeta)"

<400> 124 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 60

atccggtgca tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 120

ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaagc 180 180

agattcagca gctctggcac cggcaccgac ttcgtgttca ccatcgagaa caccctgagc 240 240

gaggacgtgg gcgactacta ctgcctgcag agctggaacg tgcccctgac ctttggcgac 300 300

ggcaccaagc tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 360

ggctctcaag tgaagctgca gcagtctggc ggaggcctcg tgaaacctgg cgcctctctg 420 420

page 82 Page 82

ggctacagcc cctacagcta cgccatggac tattggggcc agggcaccac cgtgaccgtg 720 ggctacagcc cctacagcta cgccatggac tattggggcc agggcaccao cgtgaccgtg 720

tctagtaaac ctactacaac tcctgccccc cggcctccta caccagctcc tactatcgcc 780 tctagtaaac ctactacaac tcctgccccc cggcctccta caccagctco tactatcgcc 780

tcccagccac tcagtctcag acccgaggct tctaggccag cggccggagg cgcggtccac 840 tcccagcccac tcagtctcag acccgaggct tctaggccag cggccggagg cgcggtccac 840

acccgcgggc tggactttgc atccgataag cccaccacca cccctgcccc tagacctcca 900 acccgcgggc tggactttgc atccgataag cccaccacca cccctgcccc tagacctcca 900

accccagccc ctacaatcgc cagccagccc ctgagcctga ggcccgaagc ctgtagacct 960 accccagccc ctacaatcgc cagccagccc ctgagcctga ggcccgaage ctgtagacct 960

gccgctggcg gagccgtgca caccagaggc ctggatttcg cctgcgacat ctacatctgg 1020 gccgctggcg gagccgtgca caccagaggc ctggatttcg cctgcgacat ctacatctgg 1020

gcccctctgg ccggcacctg tggcgtgctg ctgctgagcc tggtcatcac cctgtactgc 1080 gcccctctgg ccggcacctg tggcgtgctg ctgctgagcc tggtcatcac cctgtactgc 1080

aaccaccgga ataagagagg ccggaagaaa ctgctgtaca tcttcaagca gcccttcatg 1140 aaccaccgga ataagagagg ccggaagaaa ctgctgtaca tcttcaagca gcccttcatg 1140

cggcccgtgc agaccaccca ggaagaggac ggctgcagct gccggttccc cgaggaagag 1200 cggcccgtgc agaccaccca ggaagaggac ggctgcagct gccggttccc cgaggaagag 1200

gaaggcggct gcgaactgcg ggtgaagttc agccggagcg ccgacgcccc tgcctaccag 1260 gaaggcggct gcgaactgcg ggtgaagttc agccggagcg ccgacgcccc tgcctaccag 1260

cagggccaga accagctgta caacgagctg aacctgggcc ggagggagga gtacgacgtg 1320 cagggccaga accagctgta caacgagctg aacctgggcc ggagggagga gtacgacgtg 1320

ctggacaagc ggagaggccg ggaccctgag atgggcggca agccccggag aaagaaccct 1380 ctggacaagc ggagaggccg ggaccctgag atgggcggca agccccggag aaagaaccct 1380

caggagggcc tgtataacga actgcagaaa gacaagatgg ccgaggccta cagcgagatc 1440 caggagggcc tgtataacga actgcagaaa gacaagatgg ccgaggccta cagcgagato 1440

ggcatgaagg gcgagcggcg gaggggcaag ggccacgacg gcctgtacca gggcctgagc 1500 ggcatgaagg gcgagcggcg gaggggcaag ggccacgacg gcctgtacca gggcctgagc 1500

accgccacca aggataccta cgacgccctg cacatgcagg ccctgccccc caga 1554 accgccacca aggataccta cgacgccctg cacatgcagg ccctgccccc caga 1554

<210> 125 <210> 125 <211> 1695 <211> 1695 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (MR1‐1 scFv.CD8alpha 3x hinge <223> /note="EGFRvIII CAR (MR1-1 scFv. CD8alpha 3x hinge

Page 83 Page 83

50471‐706_601_SL.TXT &TM.4‐1BB.CD3‐zeta)"

(az-gW <400> 125 SCT <00 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 09

atccggtgca tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 OZI

ggcgagcccc ccaagttcct gatcagcgag ggcaacacac tgcggcctgg cgtgccaagc 180 08T

agattcagca gctctggcac cggcaccgac ttcgtgttca ccatcgagaa caccctgagc 240

gaggacgtgg gcgactacta ctgcctgcag agctggaacg tgcccctgac ctttggcgac 300 00E

ggcaccaagc tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 09E

ggctctcaag tgaagctgca gcagtctggc ggaggcctcg tgaaacctgg cgcctctctg 420

7 aagctgagct gcgtgaccag cggcttcacc ttcagaaagt tcggcatgag ctgggtgcgc 480 08/

cagaccagcg acaagcggct ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540

tactacagcg acaacgtgaa gggcagattc accatcagca gagagaacgc caagaatacc 600 009

ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccctgtacta ctgcaccaga 660 099

ggctacagcc cctacagcta cgccatggac tattggggcc agggcaccac cgtgaccgtg 720 02L

tctagtaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780 08L

agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg tgccgtccat 840

acaagaggac tggacttcgc gtccgataaa cctactacca ctccagcccc aaggccccca 900 006

accccagcac cgactatcgc atcacagcct ttgtcactgc gtcctgaagc cagccggcca 960 096

gctgcagggg gggccgtcca cacaagggga ctcgactttg cgagtgataa gcccaccacc 1020 0201

acccctgccc ctagacctcc aaccccagcc cctacaatcg ccagccagcc cctgagcctg 1080 080T

e aggcccgaag cctgtagacc tgccgctggc ggagccgtgc acaccagagg cctggatttc 1140

gcctgcgaca tctacatctg ggcccctctg gccggcacct gtggcgtgct gctgctgagc 1200 002I

ctggtcatca ccctgtactg caaccaccgg aataagagag gccggaagaa actgctgtac 1260 097T

e 9780008898 ee atcttcaagc agcccttcat gcggcccgtg cagaccaccc aggaagagga cggctgcagc 1320 OZET

tgccggttcc ccgaggaaga ggaaggcggc tgcgaactgc gggtgaagtt cagccggagc 1380 08ET

gccgacgccc ctgcctacca gcagggccag aaccagctgt acaacgagct gaacctgggc 1440

the e cggagggagg agtacgacgt gctggacaag cggagaggcc gggaccctga gatgggcggc 1500 Page 84 98 e 00ST

50471‐706_601_SL.TXT 50471-706_601_L.TXT -

aagccccgga gaaagaaccc tcaggagggc ctgtataacg aactgcagaa agacaagatg 1560 aagccccgga gaaagaaccc tcaggagggc ctgtataacg aactgcagaa agacaagatg 1560

gccgaggcct acagcgagat cggcatgaag ggcgagcggc ggaggggcaa gggccacgac 1620 gccgaggcct acagcgagat cggcatgaag ggcgagcggc ggaggggcaa gggccacgad 1620

ggcctgtacc agggcctgag caccgccacc aaggatacct acgacgccct gcacatgcag 1680 ggcctgtacc agggcctgag caccgccacc aaggatacct acgacgccct gcacatgcag 1680

gccctgcccc ccaga 1695 gccctgcccc ccaga 1695

<210> 126 <210> 126 <211> 1836 <211> 1836 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (MR1‐1 scFv.CD8alpha 4x hinge <223> /note="EGFRvIII CAR (MR1-1 scFv.CD8alpha 4x hinge &TM.4‐1BB.CD3‐zeta)" &TM.4-1BB.CD3-zeta)"

<400> 126 <400> 126 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60 gacatcgagc tgacacagag ccctgccagc ctgtctgtgg ccaccggcga gaaagtgacc 60

atccggtgca tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 atccggtgca tgaccagca cgacatcgad gacgacatga actggtatca gcagaagccc 120

ggcaccaagc tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 ggcaccaage tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360

tctagtaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780 tctagtaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780

Page 85 Page 85

50471‐706_601_SL.TXT 50471-706_601_SL.TXT agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg tgccgtccat 840 agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg tgccgtccat 840

acaagaggac tggacttcgc gtccgataaa cctactacca ctccagcccc aaggccccca 900 acaagaggac tggacttcgc gtccgataaa cctactacca ctccagcccc aaggccccca 900

accccagcac cgactatcgc atcacagcct ttgtcactgc gtcctgaagc cagccggcca 960 accccagcaa cgactatcgc atcacagcct ttgtcactgc gtcctgaagc cagccggcca 960

gctgcagggg gggccgtcca cacaagggga ctcgactttg cgagtgataa acctactaca 1020 gctgcagggg gggccgtcca cacaagggga ctcgactttg cgagtgataa acctactaca 1020

actcctgccc cccggcctcc tacaccagct cctactatcg cctcccagcc actcagtctc 1080 actcctgccc cccggcctcc tacaccagct cctactatcg cctcccagcc actcagtctc 1080

agacccgagg cttctaggcc agcggccgga ggcgcggtcc acacccgcgg gctggacttt 1140 agacccgagg cttctaggcc agcggccgga ggcgcggtcc acacccgcgg gctggacttt 1140

gcatccgata agcccaccac cacccctgcc cctagacctc caaccccagc ccctacaatc 1200 gcatccgata agcccaccao cacccctgcc cctagacctc caaccccago ccctacaatc 1200

gccagccagc ccctgagcct gaggcccgaa gcctgtagac ctgccgctgg cggagccgtg 1260 gccagccagc ccctgagcct gaggcccgaa gcctgtagac ctgccgctgg cggagccgtg 1260

cacaccagag gcctggattt cgcctgcgac atctacatct gggcccctct ggccggcacc 1320 cacaccagag gcctggattt cgcctgcgac atctacatct gggcccctct ggccggcacc 1320

tgtggcgtgc tgctgctgag cctggtcatc accctgtact gcaaccaccg gaataagaga 1380 tgtggcgtgc tgctgctgag cctggtcatc accctgtact gcaaccaccg gaataagaga 1380

ggccggaaga aactgctgta catcttcaag cagcccttca tgcggcccgt gcagaccacc 1440 ggccggaaga aactgctgta catcttcaag cagcccttca tgcggcccgt gcagaccacc 1440

caggaagagg acggctgcag ctgccggttc cccgaggaag aggaaggcgg ctgcgaactg 1500 caggaagagg acggctgcag ctgccggttc cccgaggaag aggaaggcgg ctgcgaactg 1500

cgggtgaagt tcagccggag cgccgacgcc cctgcctacc agcagggcca gaaccagctg 1560 cgggtgaagt tcagccggag cgccgacgcc cctgcctacc agcagggcca gaaccagctg 1560

tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 1620 tacaaccagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 1620

cgggaccctg agatgggcgg caagccccgg agaaagaacc ctcaggaggg cctgtataac 1680 cgggaccctg agatgggcgg caagccccgg agaaagaacc ctcaggaggg cctgtataac 1680

gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 1740 gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 1740

cggaggggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggatacc 1800 cggaggggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggatacc 1800

tacgacgccc tgcacatgca ggccctgccc cccaga 1836 tacgacgccc tgcacatgca ggccctgccc cccaga 1836

<210> 127 <210> 127 <211> 1695 <211> 1695 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (huMR1‐1 scFv.CD8alpha 3x hinge <223> /note="EGFRvIII CAR (huMR1-1 scFv. CD8alpha 3x hinge & TM.4‐1BB.CD3‐zeta)" & TM.4-1BB.CD3-zeta)

Page 86 Page 86

50471‐706_601_SL.TXT 1X1 7S <400> 127 LCT <00 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 09

atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120

the ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccagc 180 08T

agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240

gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga 300 00E

ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 09E

ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg cggaagcctg 420

7 aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag ctgggtgcgc 480 08/

cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaatacc 540

tactacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacacc 600 009

ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta ctgtgccaga 660 099

ggctacagcc cctacagcta cgccatggat tactggggcc agggcacaat ggtcaccgtg 720 022

tcctctaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780 08L

agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg tgccgtccat 840

acaagaggac tggacttcgc gtccgataaa cctactacca ctccagcccc aaggccccca 900 006

accccagcac cgactatcgc atcacagcct ttgtcactgc gtcctgaagc cagccggcca 960 096

gctgcagggg gggccgtcca cacaagggga ctcgactttg cgagtgataa gcccaccacc 1020

acccctgccc ctagacctcc aaccccagcc cctacaatcg ccagccagcc cctgagcctg 1080 080T

e aggcccgaag cctgtagacc tgccgctggc ggagccgtgc acaccagagg cctggatttc 1140

gcctgcgaca tctacatctg ggcccctctg gccggcacct gtggcgtgct gctgctgagc 1200

ctggtcatca ccctgtactg caaccaccgg aataagagag gccggaagaa actgctgtac 1260 097T

e 9780008858 ee atcttcaagc agcccttcat gcggcccgtg cagaccaccc aggaagagga cggctgcagc 1320 OZET

tgccggttcc ccgaggaaga ggaaggcggc tgcgaactgc gggtgaagtt cagccggagc 1380 08ET

gccgacgccc ctgcctacca gcagggccag aaccagctgt acaacgagct gaacctgggc 1440

cggagggagg agtacgacgt gctggacaag cggagaggcc gggaccctga gatgggcggc 1500 00ST

aagccccgga gaaagaaccc tcaggagggc ctgtataacg aactgcagaa agacaagatg 1560 09ST

eee Page 87 L8 aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

gccctgcccc ccaga 1695 gccctgcccc ccaga 1695

<210> 128 <210> 128 <211> 1836 <211> 1836 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (huMR1‐1 scFv.CD8alpha 4x hinge <223> /note="EGFRvIII CAR (huMR1-1 scFv.CD8alpha 4x hinge & TM.4‐1BB.CD3‐zeta)" & TM.4-1BB.CD3-zeta) "

<400> 128 <400> 128 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga cagagtgaco 60

atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120 atcacctgta tgaccagcac cgacatogad gacgacatga actggtatca gcagaagccc 120

agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240 agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagcco 240

ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggato tgggggaggo 360

tcctctaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780 tcctctaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780

agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg tgccgtccat 840 agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg tgccgtccat 840

Page 88 Page 88

50471‐706_601_SL.TXT 50471-706_601_SL.TXT acaagaggac tggacttcgc gtccgataaa cctactacca ctccagcccc aaggccccca 900 acaagaggad tggacttcgc gtccgataaa cctactacca ctccagcccc aaggccccca 900

accccagcac cgactatcgc atcacagcct ttgtcactgc gtcctgaagc cagccggcca 960 accccagcaa cgactatcgc atcacagcct ttgtcactgc gtcctgaago cagccggcca 960

gcatccgata agcccaccac cacccctgcc cctagacctc caaccccagc ccctacaatc 1200 gcatccgata agcccaccac cacccctgcc cctagaccto caaccccago ccctacaatc 1200

cacaccagag gcctggattt cgcctgcgac atctacatct gggcccctct ggccggcacc 1320 cacaccagag gcctggattt cgcctgcgad atctacatct gggcccctct ggccggcacc 1320

ggccggaaga aactgctgta catcttcaag cagcccttca tgcggcccgt gcagaccacc 1440 ggccggaaga aactgctgta catcttcaag cagcccttca tgcggcccgt gcagaccaco 1440

tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 1620 tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggo 1620

gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 1740 gaactgcaga aagacaagat ggcccaggcc tacagcgaga tcggcatgaa gggcgagcgg 1740

<210> 129 <210> 129 <211> 1695 <211> 1695 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (huMR1‐2 scFv.CD8alpha 3x hinge <223> /note="EGFRvIII CAR (huMR1-2 scFv. CD8alpha 3x hinge & TM.4‐1BB.CD3‐zeta)" & TM.4-1BB.CD3-zeta)" "

<400> 129 <400> 129 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcago ctgtctgcca gcgtgggcga cagagtgaco 60 Page 89 Page 89

50471‐706_601_SL.TXT 1x1'7S T09

atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca gcagaagccc 120

the ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg cgtgcccagc 180 08T

agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc cctgcagccc 240 DATE

the gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac ctttggcgga 300 00E

eee ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 09E

ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg cggaagcctg 420

aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag ctgggtgcgc 480 08/7

cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540 9799978899 STS

ttctacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacacc 600 009

ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta ctgtgccaga 660 099

ggctacagcc cctacagctt cgccatggat tactggggcc agggcacaat ggtcaccgtg 720 02L

tcctctaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780 08L

agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg tgccgtccat 840

acaagaggac tggacttcgc gtccgataaa cctactacca ctccagcccc aaggccccca 900 006

accccagcac cgactatcgc atcacagcct ttgtcactgc gtcctgaagc cagccggcca 960 096

gctgcagggg gggccgtcca cacaagggga ctcgactttg cgagtgataa gcccaccacc 1020 0201

acccctgccc ctagacctcc aaccccagcc cctacaatcg ccagccagcc cctgagcctg 1080 080T

See800088e

e aggcccgaag cctgtagacc tgccgctggc ggagccgtgc acaccagagg cctggatttc 1140

gcctgcgaca tctacatctg ggcccctctg gccggcacct gtggcgtgct gctgctgagc 1200

ctggtcatca ccctgtactg caaccaccgg aataagagag gccggaagaa actgctgtac 1260

e ee. 9780008898 092T

atcttcaagc agcccttcat gcggcccgtg cagaccaccc aggaagagga cggctgcagc 1320 OZET

tgccggttcc ccgaggaaga ggaaggcggc tgcgaactgc gggtgaagtt cagccggagc 1380 08ET esea gccgacgccc ctgcctacca gcagggccag aaccagctgt acaacgagct gaacctgggc 1440 cheese cggagggagg agtacgacgt gctggacaag cggagaggcc gggaccctga gatgggcggc 1500 00ST

aagccccgga gaaagaaccc tcaggagggc ctgtataacg aactgcagaa agacaagatg 1560 09ST

eee gccgaggcct acagcgagat cggcatgaag ggcgagcggc ggaggggcaa gggccacgac 1620 079T

Page 90 06 aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

gccctgcccc ccaga 1695 gccctgcccc ccaga 1695

<210> 130 <210> 130 <211> 1836 <211> 1836 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (huMR1‐2 scFv.CD8alpha 4x hinge <223> (note="EGFRvIII CAR (huMR1-2 scFv. CD8alpha 4x hinge & TM.4‐1BB.CD3‐zeta" & TM.4-1BB.CD3-zeta"

<400> 130 <400> 130 gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga cagagtgacc 60 gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga cagagtgacc 60

ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc tgggggaggc 360 ggcaccaagg tggaaatcaa gggcggaggo ggatctggcg gcggaggato tgggggaggo 360

cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaacacc 540 cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg ctacaacaco 540

ttctacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacacc 600 ttctacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc caagaacaco 600

ggctacagcc cctacagctt cgccatggat tactggggcc agggcacaat ggtcaccgtg 720 ggctacagcc cctacagctt cgccatggat tactggggcc agggcacaat ggtcaccgtg 720

tcctctaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780 tcctctaagc ctaccaccao ccccgcacct cgtcctccaa cccctgcacc tacgattgcc 780

Page 91 Page 91

50471‐706_601_SL.TXT 50471-706_601_SL.TXT accccagcac cgactatcgc atcacagcct ttgtcactgc gtcctgaagc cagccggcca 960 accccagcaa cgactatcgc atcacagcct ttgtcactgc gtcctgaagc cagccggcca 960

gcatccgata agcccaccac cacccctgcc cctagacctc caaccccagc ccctacaatc 1200 gcatccgata agcccaccac cacccctgcc cctagacctc caaccccago ccctacaatc 1200

tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 1620 tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 1620

<210> 131 <210> 131 <211> 3224 <211> 3224 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="XON‐64" <223> /note="XON-64"

<400> 131 <400> 131 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 60 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 60

gcaaagcatc gagcggccgc aataaaatat ctttattttc attacatctg tgtgttggtt 120 gcaaagcato gagcggccgc aataaaatat ctttattttc attacatctg tgtgttggtt 120

Page 92 Page 92

50471‐706_601_SL.TXT 1X1'7S T09 ttttgtgtga atcgtaacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac 180 08T

tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgaagga 240

tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga 300 00E

999998118e

e agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact 360 09E

gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata 420

taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc 480 08/

e tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc 540

atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt 600 009

ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt 660 099

ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct 720 OZL

acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta 780 08L

cctgagatca ccggcgaagg aggcctatca tgaagatcta tcgattgtac agctagccgc 840

the caccatgctg ctgctggtga ccagcctgct gctgtgtgag ctgccccacc ccgcctttct 900 006

gctgatcccc gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga 960 096

cagagtgacc atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca 1020 0201

the gcagaagccc ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg 1080 080T

cgtgcccagc agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc 1140

the cctgcagccc gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac 1200

ctttggcgga ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc 1260 092T

tgggggaggc ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg 1320 OZET

cggaagcctg aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag 1380 08ET

ctgggtgcgc cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg 1440

ctacaatacc tactacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc 1500 00ST

caagaacacc ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta 1560 09ST

ctgtgccaga ggctacagcc cctacagcta cgccatggat tactggggcc agggcacaat 1620 029T

ggtcaccgtg tcctctaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc 1680 089T

Page 93 E6 aged

50471‐706_601_SL.TXT 1X1 75' T09 tacgattgcc agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg 1740 DATE

tgccgtccat acaagaggac tggacttcgc gtccgataaa cctactacca ctccagcccc 1800 008T

aaggccccca accccagcac cgactatcgc atcacagcct ttgtcactgc gtcctgaagc 1860 098T

cagccggcca gctgcagggg gggccgtcca cacaagggga ctcgactttg cgagtgataa 1920 026T

gcccaccacc acccctgccc ctagacctcc aaccccagcc cctacaatcg ccagccagcc 1980 086T

e cctgagcctg aggcccgaag cctgtagacc tgccgctggc ggagccgtgc acaccagagg 2040 9702

cctggatttc gcctgcgaca tctacatctg ggcccctctg gccggcacct gtggcgtgct 2100 00I2

gctgctgagc ctggtcatca ccctgtactg caaccaccgg aataagagag gccggaagaa 2160 0912

actgctgtac atcttcaagc agcccttcat gcggcccgtg cagaccaccc aggaagagga 2220 0222

cggctgcagc tgccggttcc ccgaggaaga ggaaggcggc tgcgaactgc gggtgaagtt 2280 0822 esea cagccggagc gccgacgccc ctgcctacca gcagggccag aaccagctgt acaacgagct 2340 OTEL been gaacctgggc cggagggagg agtacgacgt gctggacaag cggagaggcc gggaccctga 2400

the gatgggcggc aagccccgga gaaagaaccc tcaggagggc ctgtataacg aactgcagaa 2460

agacaagatg gccgaggcct acagcgagat cggcatgaag ggcgagcggc ggaggggcaa 2520 0252

gggccacgac ggcctgtacc agggcctgag caccgccacc aaggatacct acgacgccct 2580 0852

gcacatgcag gccctgcccc ccagatgaga tatcactagt ctcgagtcga tcatgctcat 2640

agtggcaaga gagagccgta cgtgatcagc gggatctgct gtgccttcta gttgccagcc 2700 00LC

atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 2760 7787787078 09/2

e cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 2820

e 9997999999 0782

ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 2880 0887

tggggatgcg gtgggctcta tgggtaccca ggtgctgaag aattgacccg gttcctcctg 2940

ggccagaaag aagcaggcac atccccttct ctgtgacaca ccctgtccac gcccctggtt 3000 000E

cttagttcca gccccactca taggacactc atagctcagg agggctccgc cttcaatccc 3060 090E

acccgctaaa gtacttggag cggtctctcc ctccctcatc agcccaccaa accaaaccta 3120

gcctccaaga gtgggaagaa attaaagcaa gataggctat taagtgcaga gggagagaaa 3180 08IE

eee the atgcctccaa catgtgagga agtaatgaga gaaatcatag aatt 3224

the e Page 94 16 aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT -

<210> 132 <210> 132 <211> 3319 <211> 3319 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="XON‐30" <223> /note="XON-30"

<400> 132 <400> 132 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 60 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 60

gcaaagcatc gagcggccgc aataaaatat ctttattttc attacatctg tgtgttggtt 120 gcaaagcatc gagcggccgc aataaaatat ctttattttc attacatctg tgtgttggtt 120

ttttgtgtga atcgtaacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac 180 ttttgtgtga atcgtaacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac 180

tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgaagga 240 tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgaagga 240

tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga 300 tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga 300

agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact 360 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact 360

gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata 420 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata 420

taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc 480 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacago 480

tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc 540 tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgco 540

atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt 600 atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt 600

ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt 660 ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt 660

ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct 720 ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct 720

acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta 780 acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta 780

cctgagatca ccggcgaagg aggcctatca tgaagatcta tcgattgtac agctagccgc 840 cctgagatca ccggcgaagg aggcctatca tgaagatcta tcgattgtac agctagccgc 840

caccatgctg ctgctggtga ccagcctgct gctgtgtgag ctgccccacc ccgcctttct 900 caccatgctg ctgctggtga ccagcctgct gctgtgtgag ctgccccacc ccgcctttct 900

gctgatcccc gacatccaga tgacccagag ccccagcagc ctgtctgcca gcgtgggcga 960 gctgatcccc gacatccaga tgacccagag ccccagcage ctgtctgcca gcgtgggcga 960

cagagtgacc atcacctgta tgaccagcac cgacatcgac gacgacatga actggtatca 1020 cagagtgacc atcacctgta tgaccagcad cgacatcgad gacgacatga actggtatca 1020

gcagaagccc ggcaagaccc ccaagctgct gatctacgag ggcaacaccc tgaggcctgg 1080 gcagaagccc ggcaagacco ccaagctgct gatctacgag ggcaacaccc tgaggcctgg 1080

Page 95 Page 95

50471‐706_601_SL.TXT cgtgcccagc agattttctg gcagcggctc cggcaccgac ttcatcttca ccatcagctc 1140

cctgcagccc gaggatatcg ccacctacta ctgcctgcag agctggaacg tgcccctgac 1200

ctttggcgga ggcaccaagg tggaaatcaa gggcggaggc ggatctggcg gcggaggatc 1260 092T

tgggggaggc ggctctcagg tgcagctgca ggaatctggc ggagggctcg tgaagcctgg 1320 OZET

e cggaagcctg aagctgagct gtgccgccag cggcttcacc ttcagcaagt tcggcatgag 1380 08ET

ctgggtgcgc cagacccccg acaagagact ggaatgggtg gccagcatca gcaccggcgg 1440

ctacaatacc tactacagcg acaacgtgaa gggccggttc accatctccc gggacaacgc 1500 00ST

I caagaacacc ctgtacctgc agatgagcag cctgaagtcc gaggacaccg ccatgtacta 1560 09ST

ctgtgccaga ggctacagcc cctacagcta cgccatggat tactggggcc agggcacaat 1620 029T

ggtcaccgtg tcctctaagc ctaccaccac ccccgcacct cgtcctccaa cccctgcacc 1680 089T

tacgattgcc agtcagcctc tttcactgcg gcctgaggcc agcagaccag ctgccggcgg 1740

tgccgtccat acaagaggac tggacttcgc gtccgataaa cctactacca ctccagcccc 1800 008T

aaggccccca accccagcac cgactatcgc atcacagcct ttgtcactgc gtcctgaagc 1860 098T

cagccggcca gctgcagggg gggccgtcca cacaagggga ctcgactttg cgagtgataa 1920 026T

gcccaccacc acccctgccc ctagacctcc aaccccagcc cctacaatcg ccagccagcc 1980 086T

e cctgagcctg aggcccgaag cctgtagacc tgccgctggc ggagccgtgc acaccagagg 2040

eee ee. 9780008808 9702

cctggatttc gcctgcgaca tctacatctg ggcccctctg gccggcacct gtggcgtgct 2100 0012

gctgctgagc ctggtcatca ccctgtactg caaccaccgg aataagagag gccggaagaa 2160 0912

actgctgtac atcttcaagc agcccttcat gcggcccgtg cagaccaccc aggaagagga 2220 0222

cggctgcagc tgccggttcc ccgaggaaga ggaaggcggc tgcgaactgc gggtgaagtt 2280 0822

cagccggagc gccgacgccc ctgcctacca gcagggccag aaccagctgt acaacgagct 2340 OTEC

e gaacctgggc cggagggagg agtacgacgt gctggacaag cggagaggcc gggaccctga 2400

gatgggcggc aagccccgga gaaagaaccc tcaggagggc ctgtataacg aactgcagaa 2460

eee agacaagatg gccgaggcct acagcgagat cggcatgaag ggcgagcggc ggaggggcaa 2520 0252

gggccacgac ggcctgtacc agggcctgag caccgccacc aaggatacct acgacgccct 2580 0857

gcacatgcag gccctgcccc ccagaagggc caagaggagt ggcagcggcg agggcagagg 2640

Page 96 96 aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT aagtcttcta acatgcggtg acgtggagga gaatcccggc cctatgaggc tccctgctca aagtcttcta acatgcggtg acgtggagga gaatcccggc cctatgaggc tccctgctca 2700 2700

gctcctgggg ctgctaatgc tctgggtccc aggatccagt gggcgcaaag tgtgtaacgg gctcctgggg ctgctaatgc tctgggtccc aggatccagt gggcgcaaag tgtgtaacgg 2760 2760

aataggtatt ggtgaattta aagactcact ctccataaat gctacgaata ttaaacactt aataggtatt ggtgaattta aagactcact ctccataaat gctacgaata ttaaacactt 2820 2820

caaaaactgc acctccatca gtggcgatct ccacatcctg ccggtggcat ttaggggtga caaaaactgc acctccatca gtggcgatct ccacatcctg ccggtggcat ttaggggtga 2880 2880

ctccttcaca catactcctc ctctggatcc acaggaactg gatattctga aaaccgtaaa ctccttcaca catactcctc ctctggatcc acaggaactg gatattctga aaaccgtaaa 2940 2940

ggaaatcaca gggtttttgc tgattcaggc ttggcctgaa aacaggacgg acctccatgc ggaaatcaca gggtttttgc tgattcaggc ttggcctgaa aacaggacgg acctccatgc 3000 3000

ctttgagaac ctagaaatca tacgcggcag gaccaagcaa catggtcagt tttctcttgc ctttgagaac ctagaaatca tacgcggcag gaccaagcaa catggtcagt tttctcttgc 3060 3060

agtcgtcagc ctgaacataa catccttggg attacgctcc ctcaaggaga taagtgatgg agtcgtcagc ctgaacataa catccttggg attacgctcc ctcaaggaga taagtgatgg 3120 3120

agatgtgata atttcaggaa acaaaaattt gtgctatgca aatacaataa actggaaaaa agatgtgata atttcaggaa acaaaaattt gtgctatgca aatacaataa actggaaaaa 3180 3180

actgtttggg acctccggtc agaaaaccaa aattataagc aacagaggtg aaaacagctg actgtttggg acctccggtc agaaaaccaa aattataagc aacagaggtg aaaacagctg 3240 3240

caaggccaca ggccaggtct gccatgcctt gtgctccccc gagggctgct ggggcccgga 3300 caaggccaca ggccaggtct gccatgcctt gtgctccccc gagggctgct ggggcccgga 3300

gcccagggac tgcgtctct 3319 gcccagggad tgcgtctct 3319

<210> 133 <210> 133 <211> 4763 <211> 4763 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note= "Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="XON‐59" <223> /note="XON-59" -

<400> 133 <400> 133 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagago gcacatcgcc cacagtcccc gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60 60

gagaagttgg ggggagggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt gagaagttgg ggggaggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt 120 120

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 180

gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240 240

acagcagccg ctaaatccaa ggtaaggtca gaagagctag cgccaccatg ctgctgctgg 300 acagcagccg ctaaatccaa ggtaaggtca gaagagctag cgccaccatg ctgctgctgg 300

tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatcc tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatcc 360 360

Page 97 Page 97

50471‐706_601_SL.TXT agatgaccca gagccccagc agcctgtctg ccagcgtggg cgacagagtg accatcacct 420

7 gtatgaccag caccgacatc gacgacgaca tgaactggta tcagcagaag cccggcaaga 480 08/

eee cccccaagct gctgatctac gagggcaaca ccctgaggcc tggcgtgccc agcagatttt 540

ctggcagcgg ctccggcacc gacttcatct tcaccatcag ctccctgcag cccgaggata 600 009

tcgccaccta ctactgcctg cagagctgga acgtgcccct gacctttggc ggaggcacca 660 099

aggtggaaat caagggcgga ggcggatctg gcggcggagg atctggggga ggcggctctc 720 OZL

aggtgcagct gcaggaatct ggcggagggc tcgtgaagcc tggcggaagc ctgaagctga 780 08L

gctgtgccgc cagcggcttc accttcagca agttcggcat gagctgggtg cgccagaccc 840

ccgacaagag actggaatgg gtggccagca tcagcaccgg cggctacaat acctactaca 900 006

gcgacaacgt gaagggccgg ttcaccatct cccgggacaa cgccaagaac accctgtacc 960 096

tgcagatgag cagcctgaag tccgaggaca ccgccatgta ctactgtgcc agaggctaca 1020 0201

gcccctacag ctacgccatg gattactggg gccagggcac aatggtcacc gtgtcctcta 1080 080I

agcctaccac cacccccgca cctcgtcctc caacccctgc acctacgatt gccagtcagc 1140

ctctttcact gcggcctgag gccagcagac cagctgccgg cggtgccgtc catacaagag 1200

gactggactt cgcgtccgat aaacctacta ccactccagc cccaaggccc ccaaccccag 1260 092T

caccgactat cgcatcacag cctttgtcac tgcgtcctga agccagccgg ccagctgcag 1320 OZET

ggggggccgt ccacacaagg ggactcgact ttgcgagtga taagcccacc accacccctg 1380 08ET

cccctagacc tccaacccca gcccctacaa tcgccagcca gcccctgagc ctgaggcccg 1440

aagcctgtag acctgccgct ggcggagccg tgcacaccag aggcctggat ttcgcctgcg 1500 00ST

acatctacat ctgggcccct ctggccggca cctgtggcgt gctgctgctg agcctggtca 1560 09ST

tcaccctgta ctgcaaccac cggaataaga gaggccggaa gaaactgctg tacatcttca 1620

agcagccctt catgcggccc gtgcagacca cccaggaaga ggacggctgc agctgccggt 1680 089T

eee tccccgagga agaggaaggc ggctgcgaac tgcgggtgaa gttcagccgg agcgccgacg 1740

cccctgccta ccagcagggc cagaaccagc tgtacaacga gctgaacctg ggccggaggg 1800 008I

aggagtacga cgtgctggac aagcggagag gccgggaccc tgagatgggc ggcaagcccc 1860 098T

ggagaaagaa ccctcaggag ggcctgtata acgaactgca gaaagacaag atggccgagg 1920 026T

Seededeee8 ee. Page 98 86 aged

50471‐706_601_SL.TXT cctacagcga gatcggcatg aagggcgagc ggcggagggg caagggccac gacggcctgt 1980 9999.99.88 086T

accagggcct gagcaccgcc accaaggata cctacgacgc cctgcacatg caggccctgc 2040 9702

cccccagaag agctaagagg ggaagcggag agggcagagg aagtctgcta acatgcggtg 2100 0012

acgtcgagga gaatcctgga cctggcccca agaagaaaag gaaggtggcc ccccccaccg 2160 0912

acgtgagcct gggcgacgag ctgcacctgg acggcgagga cgtggccatg gcccacgccg 2220 0222

acgccctgga cgacttcgac ctggacatgc tgggcgacgg cgacagcccc ggccccggct 2280 0877

e tcacccccca cgacagcgcc ccctacggcg ccctggacat ggccgacttc gagttcgagc 2340

agatgttcac cgacgccctg ggcatcgacg agtacggcgg cgaattcgag atgcccgtgg 2400

acaggattct ggaggccgaa ctcgccgtgg agcagaaaag cgaccagggc gtggagggcc 2460

ccggcggaac cggcggcagc ggcagcagcc ccaacgaccc cgtgaccaac atctgccagg 2520 0252

ccgccgacaa gcagctgttc accctggtgg agtgggccaa gaggattccc cacttcagca 2580 0857

ee gcctgcccct ggacgaccag gtgatcctgc tgagggccgg atggaacgag ctgctgatcg 2640

ccagcttcag ccacaggagc atcgacgtga gggacggcat cctgctggcc accggcctgc 2700 00/2

acgtccatag gaacagcgcc cacagcgccg gagtgggcgc catcttcgac agggtgctga 2760 09/2

ccgagctggt gagcaagatg agggacatga ggatggacaa gaccgagctg ggctgcctga 2820 0787

gggccatcat cctgttcaac cccgaggtga ggggcctgaa aagcgcccag gaggtggagc 2880 0887

tgctgaggga gaaggtgtac gccgccctgg aggagtacac caggaccacc caccccgacg 2940 9767

agcccggcag attcgccaag ctgctgctga ggctgcccag cctgaggagc atcggcctga 3000 000E

The agtgcctgga gcacctgttc ttcttcaggc tgatcggcga cgtgcccatc gacaccttcc 3060 090E

tgatggagat gctggagagc cccagcgaca gcagagctaa gaggggaagc ggagagggca 3120 OTTE

gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggacctaag ctgctgagca 3180 08TE

gcatcgagca ggcttgcgac atctgcaggc tgaagaagct gaagtgcagc aaggagaagc 3240

ccaagtgcgc caagtgcctg aagaacaact gggagtgcag atacagcccc aagaccaaga 3300 00EE

ggagccccct gaccagggcc cacctgaccg aggtggagag caggctggag aggctggagc 3360 09EE

e agctgttcct gctgatcttc cccagggagg acctggacat gatcctgaag atggacagcc 3420 000000000

tgcaagacat caaggccctg ctgaccggcc tgttcgtgca ggacaacgtg aacaaggacg 3480

Page 99 66 anded

50471‐706_601_SL.TXT 50471-706_601_SL.TXT ccgtgaccga caggctggcc agcgtggaga ccgacatgcc cctgaccctg aggcagcaca 3540 ccgtgaccga caggctggcc agcgtggaga ccgacatgcc cctgaccctg aggcagcaca 3540

ggatcagcgc caccagcagc agcgaggaga gcagcaacaa gggccagagg cagctgaccg 3600 ggatcagcgc caccagcage agcgaggaga gcagcaacaa gggccagagg cagctgaccg 3600

tgagccccga gtttcccggg atcaggcccg agtgcgtggt gcccgagacc cagtgcgcca 3660 tgagccccga gtttcccggg atcaggcccg agtgcgtggt gcccgagacc cagtgcgcca 3660

tgaaaaggaa ggagaagaag gcccagaagg agaaggacaa gctgcccgtg agcaccacca 3720 tgaaaaggaa ggagaagaag gcccagaagg agaaggacaa gctgcccgtg agcaccacca 3720

ccgtcgatga ccacatgccc cccatcatgc agtgcgagcc cccccccccc gaggccgcca 3780 ccgtcgatga ccacatgccc cccatcatgc agtgcgagcc CCCCCCCCCCC gaggccgcca 3780

ggattcacga ggtcgtgccc aggttcctga gcgacaagct gctggtgacc aacaggcaga 3840 ggattcacga ggtcgtgccc aggttcctga gcgacaagct gctggtgacc aacaggcaga 3840

agaacatccc ccagctgacc gccaaccagc agttcctgat cgccaggctg atctggtatc 3900 agaacatccc ccagctgacc gccaaccago agttcctgat cgccaggctg atctggtatc 3900

aggacggcta cgagcagccc agcgacgagg acctgaaaag gatcacccag acctggcagc 3960 aggacggcta cgagcagccc agcgacgagg acctgaaaag gatcacccag acctggcagc 3960

aggccgacga cgagaacgag gagagcgaca cccccttcag gcagatcacc gagatgacca 4020 aggccgacga cgagaacgag gagagcgaca cccccttcag gcagatcacc gagatgacca 4020

tcctgaccgt gcagctgatc gtggagttcg ccaagggcct gcccggattc gccaagatca 4080 tcctgaccgt gcagctgato gtggagttcg ccaagggcct gcccggatto gccaagatca 4080

gccagcccga ccagatcacc ctgctgaagg cttgcagcag cgaggtgatg atgctgaggg 4140 gccagcccga ccagatcacc ctgctgaagg cttgcagcag cgaggtgatg atgctgaggg 4140

tggccaggag gtacgacgcc gccagcgaca gcatcctgtt cgccaacaac caggcttaca 4200 tggccaggag gtacgacgcc gccagcgaca gcatcctgtt cgccaacaac caggettaca 4200

ccagggacaa ctacaggaag gctggcatgg ccgaggtgat cgaggacctc ctgcacttct 4260 ccagggacaa ctacaggaag gctggcatgg ccgaggtgat cgaggacctc ctgcacttct 4260

gcagatgtat gtacagcatg gccctggaca acatccacta cgccctgctg accgccgtgg 4320 gcagatgtat gtacagcatg gccctggaca acatccacta cgccctgctg accgccgtgg 4320

tgatcttcag cgacaggccc ggcctggagc agccccagct ggtggaggag atccagaggt 4380 tgatcttcag cgacaggcco ggcctggagc agccccagct ggtggaggag atccagaggt 4380

actacctgaa caccctgagg atctacatcc tgaaccagct gagcggcagc gccaggagca 4440 actacctgaa caccctgagg atctacatcc tgaaccagct gagcggcagc gccaggagca 4440

gcgtgatcta cggcaagatc ctgagcatcc tgagcgagct gaggaccctg ggaatgcaga 4500 gcgtgatcta cggcaagatc ctgagcatcc tgagcgagct gaggaccctg ggaatgcaga 4500

acagcaatat gtgtatcagc ctgaagctga agaacaggaa gctgcccccc ttcctggagg 4560 acagcaatat gtgtatcagc ctgaagctga agaacaggaa gctgcccccc ttcctggagg 4560

agatttggga cgtggccgac atgagccaca cccagccccc ccccatcctg gagagcccca 4620 agatttggga cgtggccgac atgagccaca cccagccccc ccccatcctg gagagcccca 4620

ccaacctgtg aaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 4680 ccaacctgtg aaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 4680

caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 4740 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 4740

tcaatgtatc ttatcatgtc tgg 4763 tcaatgtatc ttatcatgtc tgg 4763

<210> 134 <210> 134 <211> 4785 <211> 4785 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source

Page 100 Page 100

50471‐706_601_SL.TXT 50471-706_601_SLTXT <223> /note="Description of Artificial Sequence: Synthetic <223> /note= 'Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="XON‐60" <223> /note="XON-60"

<400> 134 <400> 134 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60

gagaagttgg ggggaggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt 120 gagaagttgg ggggagggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt 120

tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatcc 360 tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatcc 360

agatgaccca gagccccagc agcctgtctg ccagcgtggg cgacagagtg accatcacct 420 agatgaccca gagccccagc agcctgtctg ccagcgtggg cgacagagtg accatcacct 420

gtatgaccag caccgacatc gacgacgaca tgaactggta tcagcagaag cccggcaaga 480 gtatgaccag caccgacato gacgacgaca tgaactggta tcagcagaag cccggcaaga 480

cccccaagct gctgatctac gagggcaaca ccctgaggcc tggcgtgccc agcagatttt 540 cccccaagct gctgatctac gagggcaaca ccctgaggcc tggcgtgccc agcagatttt 540

ctggcagcgg ctccggcacc gacttcatct tcaccatcag ctccctgcag cccgaggata 600 ctggcagcgg ctccggcacc gacttcatct tcaccatcag ctccctgcag cccgaggata 600

tcgccaccta ctactgcctg cagagctgga acgtgcccct gacctttggc ggaggcacca 660 tcgccaccta ctactgcctg cagagctgga acgtgcccct gacctttggc ggaggcacca 660

aggtggaaat caagggcgga ggcggatctg gcggcggagg atctggggga ggcggctctc 720 aggtggaaat caagggcgga ggcggatctg gcggcggagg atctggggga ggcggctctc 720

aggtgcagct gcaggaatct ggcggagggc tcgtgaagcc tggcggaagc ctgaagctga 780 aggtgcagct gcaggaatct ggcggagggc tcgtgaagcc tggcggaagc ctgaagctga 780

gctgtgccgc cagcggcttc accttcagca agttcggcat gagctgggtg cgccagaccc 840 gctgtgccgc cagcggcttc accttcagca agttcggcat gagctgggtg cgccagaccc 840

ccgacaagag actggaatgg gtggccagca tcagcaccgg cggctacaat acctactaca 900 ccgacaagag actggaatgg gtggccagca tcagcaccgg cggctacaat acctactaca 900

gcgacaacgt gaagggccgg ttcaccatct cccgggacaa cgccaagaac accctgtacc 960 gcgacaacgt gaagggccgg ttcaccatct cccgggacaa cgccaagaac accctgtacc 960

tgcagatgag cagcctgaag tccgaggaca ccgccatgta ctactgtgcc agaggctaca 1020 tgcagatgag cagcctgaag tccgaggaca ccgccatgta ctactgtgcc agaggctaca 1020

gcccctacag ctacgccatg gattactggg gccagggcac aatggtcacc gtgtcctcta 1080 gcccctacag ctacgccatg gattactggg gccagggcac aatggtcacc gtgtcctcta 1080

agcctaccac cacccccgca cctcgtcctc caacccctgc acctacgatt gccagtcagc 1140 agcctaccac cacccccgca cctcgtcctc caacccctgc acctacgatt gccagtcago 1140

ctctttcact gcggcctgag gccagcagac cagctgccgg cggtgccgtc catacaagag 1200 ctctttcact gcggcctgag gccagcagac cagctgccgg cggtgccgtc catacaagag 1200

gactggactt cgcgtccgat aaacctacta ccactccagc cccaaggccc ccaaccccag 1260 gactggactt cgcgtccgat aaacctacta ccactccago cccaaggccc ccaaccccag 1260

caccgactat cgcatcacag cctttgtcac tgcgtcctga agccagccgg ccagctgcag 1320 caccgactat cgcatcacag cctttgtcac tgcgtcctga agccagccgg ccagctgcag 1320

Page 101 Page 101

50471‐706_601_SL.TXT ggggggccgt ccacacaagg ggactcgact ttgcgagtga taagcccacc accacccctg 1380 08EI

cccctagacc tccaacccca gcccctacaa tcgccagcca gcccctgagc ctgaggcccg 1440

aagcctgtag acctgccgct ggcggagccg tgcacaccag aggcctggat ttcgcctgcg 1500 00ST

acatctacat ctgggcccct ctggccggca cctgtggcgt gctgctgctg agcctggtca 1560 09ST

tcaccctgta ctgcaaccac cggaataaga gaggccggaa gaaactgctg tacatcttca 1620 0291

agcagccctt catgcggccc gtgcagacca cccaggaaga ggacggctgc agctgccggt 1680 089T

tccccgagga agaggaaggc ggctgcgaac tgcgggtgaa gttcagccgg agcgccgacg 1740

e cccctgccta ccagcagggc cagaaccagc tgtacaacga gctgaacctg ggccggaggg 1800 008T

aggagtacga cgtgctggac aagcggagag gccgggaccc tgagatgggc ggcaagcccc 1860 0000800088 098T

ggagaaagaa ccctcaggag ggcctgtata acgaactgca gaaagacaag atggccgagg 1920 Seededeee8 eeGeee8e88 026T

cctacagcga gatcggcatg aagggcgagc ggcggagggg caagggccac gacggcctgt 1980 086T

been accagggcct gagcaccgcc accaaggata cctacgacgc cctgcacatg caggccctgc 2040 9702

cccccagaag agctaagagg ggaagcggag agggcagagg aagtctgcta acatgcggtg 2100 0012

acgtcgagga gaatcctgga cctggcccca agaagaaaag gaaggtggcc ccccccaccg 2160 0912

acgtgagcct gggcgacgag ctgcacctgg acggcgagga cgtggccatg gcccacgccg 2220 0222

e acgccctgga cgacttcgac ctggacatgc tgggcgacgg cgacagcccc ggccccggct 2280

e 0877

tcacccccca cgacagcgcc ccctacggcg ccctggacat ggccgacttc gagttcgagc 2340 OTEL

agatgttcac cgacgccctg ggcatcgacg agtacggcgg cgaattcgag atgcccgtgg 2400 997800087e

acaggattct ggaggccgaa ctcgccgtgg agcagaaaag cgaccagggc gtggagggcc 2460

ccggcggaac cggcggcagc ggcagcagcc ccaacgaccc cgtgaccaac atctgccagg 2520 0252

ccgccgacaa gcagctgttc accctggtgg agtgggccaa gaggattccc cacttcagca 2580 0857

ee gcctgcccct ggacgaccag gtgatcctgc tgagggccgg atggaacgag ctgctgatcg 2640

ccagcttcag ccacaggagc atcgacgtga gggacggcat cctgctggcc accggcctgc 2700 00/2

acgtccatag gaacagcgcc cacagcgccg gagtgggcgc catcttcgac agggtgctga 2760 09/2

ccgagctggt gagcaagatg agggacatga ggatggacaa gaccgagctg ggctgcctga 2820 0787

gggccatcat cctgttcaac cccgaggtga ggggcctgaa aagcgcccag gaggtggagc 2880 0887

Page 102 ZOT aged See

50471‐706_601_SL.TXT I09 tgctgaggga gaaggtgtac gccgccctgg aggagtacac caggaccacc caccccgacg 2940 9767

agcccggcag attcgccaag ctgctgctga ggctgcccag cctgaggagc atcggcctga 3000 000E

agtgcctgga gcacctgttc ttcttcaggc tgatcggcga cgtgcccatc gacaccttcc 3060 090E

tgatggagat gctggagagc cccagcgaca gctgagcatg cactagtttt ataatttctt 3120 OZIE

cttccagaat ttctgacatt ttataatttc ttcttccaga agactcacaa cctccatatg 3180 08TE

gccaccatga agctgctgag cagcatcgag caggcttgcg acatctgcag gctgaagaag 3240

ctgaagtgca gcaaggagaa gcccaagtgc gccaagtgcc tgaagaacaa ctgggagtgc 3300 00EE

ee agatacagcc ccaagaccaa gaggagcccc ctgaccaggg cccacctgac cgaggtggag 3360 09EE

agcaggctgg agaggctgga gcagctgttc ctgctgatct tccccaggga ggacctggac 3420

eee atgatcctga agatggacag cctgcaagac atcaaggccc tgctgaccgg cctgttcgtg 3480 9780778700

caggacaacg tgaacaagga cgccgtgacc gacaggctgg ccagcgtgga gaccgacatg 3540

cccctgaccc tgaggcagca caggatcagc gccaccagca gcagcgagga gagcagcaac 3600 009E

been e aagggccaga ggcagctgac cgtgagcccc gagtttcccg ggatcaggcc cgagtgcgtg 3660 099 gtgcccgaga cccagtgcgc catgaaaagg aaggagaaga aggcccagaa ggagaaggac 3720 OZLE beddee8e88 ee edeededdee

e aagctgcccg tgagcaccac caccgtcgat gaccacatgc cccccatcat gcagtgcgag 3780

0878078899 0000000000 08LE

cccccccccc ccgaggccgc caggattcac gaggtcgtgc ccaggttcct gagcgacaag 3840

ctgctggtga ccaacaggca gaagaacatc ccccagctga ccgccaacca gcagttcctg 3900 0068

atcgccaggc tgatctggta tcaggacggc tacgagcagc ccagcgacga ggacctgaaa 3960 0968

aggatcaccc agacctggca gcaggccgac gacgagaacg aggagagcga cacccccttc 4020

e. aggcagatca ccgagatgac catcctgacc gtgcagctga tcgtggagtt cgccaagggc 4080 080t

ctgcccggat tcgccaagat cagccagccc gaccagatca ccctgctgaa ggcttgcagc 4140

agcgaggtga tgatgctgag ggtggccagg aggtacgacg ccgccagcga cagcatcctg 4200

7 ttcgccaaca accaggctta caccagggac aactacagga aggctggcat ggccgaggtg 4260

atcgaggacc tcctgcactt ctgcagatgt atgtacagca tggccctgga caacatccac 4320

tacgccctgc tgaccgccgt ggtgatcttc agcgacaggc ccggcctgga gcagccccag 4380

ctggtggagg agatccagag gtactacctg aacaccctga ggatctacat cctgaaccag 4440

Page 103 EOT aged 2

50471‐706_601_SL.TXT 50471-706_601_SL.TXT ctgagcggca gcgccaggag cagcgtgatc tacggcaaga tcctgagcat cctgagcgag ctgagcggca gcgccaggag cagcgtgatc tacggcaaga tcctgagcat cctgagcgag 4500 4500 ctgaggacco tgggaatgca gaacagcaat atgtgtatca gcctgaagct gaagaacagg ctgaggaccc tgggaatgca gaacagcaat atgtgtatca gcctgaagct gaagaacagg 4560 4560 aagctgcccc ccttcctgga ggagatttgg gacgtggccg acatgagcca cacccagccc aagctgcccc ccttcctgga ggagatttgg gacgtggccg acatgagcca cacccagccc 4620 4620 ccccccatcc tggagagccc caccaacctg tgaaacttgt ttattgcagc ttataatggt ccccccatcc tggagagccc caccaacctg tgaaacttgt ttattgcagc ttataatggt 4680 4680 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 4740 4740 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgg agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgg 4785 4785

<210> 135 <210> 135 <211> 2009 <211> 2009 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="XON‐61" <223> /note="XON-61" -

<400> 135 <400> 135 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60 60 gactattccg gggtttaccg gcgcactctc gcccgaactt caccggcggt ctttcgtccg gactattccg gggtttaccg gcgcactctc gcccgaactt caccggcggt ctttcgtccg 120 120 tgctttatcg gggcggatca ctccgaaccc cgggaggtct atataagcag agctcgttta tgctttatcg gggcggatca ctccgaaccc cgggaggtct atataagcag agctcgttta 180 180 gtgaaccctc attctggaga cggatcccga gccgagtgtt ttgacctcca tagaacagco gtgaaccctc attctggaga cggatcccga gccgagtgtt ttgacctcca tagaacagcc 240 240 gctaaatcca aggtaaggto agaagagcta gcgccaccat gtgtcaccag cagttggtca gctaaatcca aggtaaggtc agaagagcta gcgccaccat gtgtcaccag cagttggtca 300 300 tctcttggtt cagcctggtt tttctggcat ctcccctcgt ggccatctgg gaactgaaga tctcttggtt cagcctggtt tttctggcat ctcccctcgt ggccatctgg gaactgaaga 360 360

aagatgttta tgtcgtagaa ttggattggt atcccgacgc ccctggagaa atggtggtcc aagatgttta tgtcgtagaa ttggattggt atcccgacgc ccctggagaa atggtggtcc 420 420 tgacatgtga cacccctgaa gaagatggta tcacctggac cttggaccag agcagtgagg tgacatgtga cacccctgaa gaagatggta tcacctggac cttggaccag agcagtgagg 480 480 tcttaggctc tggcaagacc ctgaccatcc aagtcaaaga gtttggagat gctggccagt tcttaggctc tggcaagacc ctgaccatcc aagtcaaaga gtttggagat gctggccagt 540 540

acacctgtca caaaggaggo gaggttctaa gccattcgct cctgctgctt cacaaaaagg acacctgtca caaaggaggc gaggttctaa gccattcgct cctgctgctt cacaaaaagg 600 600 aagatggaat ttggtccact gacattctga aggaccagaa agaacccaag aataagacct aagatggaat ttggtccact gacattctga aggaccagaa agaacccaag aataagacct 660 660 ttctaagatg cgaggccaag aattattctg gacgtttcad ctgctggtgg ctgacgacaa ttctaagatg cgaggccaag aattattctg gacgtttcac ctgctggtgg ctgacgacaa 720 720

Page 104 Page 104

50471-706_601_SL.TXT 50471‐706_601_SL.TXT tcagtactga tttgacattc agtgtcaaaa gcagcagagg ctcttctgac ccccaagggg tcagtactga tttgacattc agtgtcaaaa gcagcagagg ctcttctgac ccccaagggg 780 780 tgacgtgcgg agctgctaca ctcagcgccg agagagtcag aggggacaac aaggagtatg tgacgtgcgg agctgctaca ctcagcgccg agagagtcag aggggacaac aaggagtatg 840 840 agtactcagt ggagtgccag gaggacagtg cctgcccagc tgctgaggag agtctgccca agtactcagt ggagtgccag gaggacagtg cctgcccagc tgctgaggag agtctgccca 900 900 ttgaggtcat ggtggatgcc gttcacaagc tcaagtatga aaactacacc agcagcttct ttgaggtcat ggtggatgcc gttcacaagc tcaagtatga aaactacacc agcagcttct 960 960 tcatcaggga catcatcaaa cctgacccac ccaagaactt gcagctgaag cccctgaaga tcatcaggga catcatcaaa cctgacccac ccaagaactt gcagctgaag cccctgaaga 1020 1020 acagcagaca ggtggaggtc agctgggagt accctgacac ctggagtact ccacattcct acagcagaca ggtggaggtc agctgggagt accctgacac ctggagtact ccacattcct 1080 1080 acttctccct gacattctgc gttcaggtcc agggcaagag caagagagaa aagaaagata acttctccct gacattctgc gttcaggtcc agggcaagag caagagagaa aagaaagata 1140 1140 gagtcttcac ggacaagacc tcagccacgg tcatctgccg caaaaatgcc agcattagcg gagtcttcac ggacaagacc tcagccacgg tcatctgccg caaaaatgcc agcattagcg 1200 1200 tgcgggccca ggaccgctac tatagctcat cttggagcga atgggcatct gtgccctgct tgcgggccca ggaccgctac tatagctcat cttggagcga atgggcatct gtgccctgct 1260 1260 ccggtggcgg tggcggcgga tctagaaacc tccccgtggc cactccagac ccaggaatgt ccggtggcgg tggcggcgga tctagaaacc tccccgtggc cactccagac ccaggaatgt 1320 1320 tcccatgcct tcaccacage cagaacctgc tgagggccgt cagcaacatg ctccagaagg tcccatgcct tcaccacagc cagaacctgc tgagggccgt cagcaacatg ctccagaagg 1380 1380 ccagacaaac tctagaattt tacccttgca cttctgaaga gattgatcat gaagatatca ccagacaaac tctagaattt tacccttgca cttctgaaga gattgatcat gaagatatca 1440 1440 caaaagataa aaccagcaca gtggaggcct gtttaccatt ggaattaacc aagaatgaga caaaagataa aaccagcaca gtggaggcct gtttaccatt ggaattaacc aagaatgaga 1500 1500 gttgcctaaa ttccagagag acctctttca taactaatgg gagttgcctg gcctccagaa gttgcctaaa ttccagagag acctctttca taactaatgg gagttgcctg gcctccagaa 1560 1560 agacctcttt tatgatggcc ctgtgcctta gtagtattta tgaagacttg aagatgtacc agacctcttt tatgatggcc ctgtgcctta gtagtattta tgaagacttg aagatgtacc 1620 1620 aggtggagtt caagaccatg aatgcaaagc tgctgatgga ccccaagagg cagatctttc aggtggagtt caagaccatg aatgcaaagc tgctgatgga ccccaagagg cagatctttc 1680 1680 tagatcaaaa catgctggca gttattgatg agctgatgca ggccctgaat ttcaacagtg tagatcaaaa catgctggca gttattgatg agctgatgca ggccctgaat ttcaacagtg 1740 1740 agactgtgcc acaaaaatcc tcccttgaag aaccggattt ttataaaact aaaatcaagc agactgtgcc acaaaaatcc tcccttgaag aaccggattt ttataaaact aaaatcaagc 1800 1800 tctgcatact tcttcatgct ttcagaatca gagcagtgac tattgataga gtgatgagct tctgcatact tcttcatgct ttcagaatca gagcagtgac tattgataga gtgatgagct 1860 1860 atctgaatgc ttcctaaaac ttgtttattg cagcttataa tggttacaaa taaagcaata atctgaatgc ttcctaaaac ttgtttattg cagcttataa tggttacaaa taaagcaata 1920 1920 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 1980 1980

aactcatcaa tgtatcttat catgtctgg aactcatcaa tgtatcttat catgtctgg 2009 2009

<210> 136 <210> 136 <211> 2873 <211> 2873 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source Page 105 Page 105

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <223> /note= "Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="XON‐62" <223> /note="XON-62"

<400> 136 <400> 136 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60 60

gactattccg gggtttaccg gcgcactctc gcccgaactt caccggcggt ctttcgtccg gactattccg gggtttaccg gcgcactctc gcccgaactt caccggcggt ctttcgtccg 120 120

tgctttatcg gggcggatca ctccgaacco cgggaggtct atataagcag agctcgttta tgctttatcg gggcggatca ctccgaaccc cgggaggtct atataagcag agctcgttta 180 180

gtgaaccctc attctggaga cggatcccga gccgagtgtt ttgacctcca tagaacagcc gtgaaccctc attctggaga cggatcccga gccgagtgtt ttgacctcca tagaacagcc 240 240

gctaaatcca aggtaaggtc agaagagcta gcgccaccat gtgtcaccag cagttggtca gctaaatcca aggtaaggtc agaagagcta gcgccaccat gtgtcaccag cagttggtca 300 300

tctcttggtt cagcctggtt tttctggcat ctcccctcgt ggccatctgg gaactgaaga tctcttggtt cagcctggtt tttctggcat ctcccctcgt ggccatctgg gaactgaaga 360 360

acacctgtca caaaggaggo gaggttctaa gccattcgct cctgctgctt cacaaaaagg acacctgtca caaaggaggc gaggttctaa gccattcgct cctgctgctt cacaaaaagg 600 600

aagatggaat ttggtccact gacattctga aggaccagaa agaacccaag aataagacct aagatggaat ttggtccact gacattctga aggaccagaa agaacccaag aataagacct 660 660 ttctaagatg cgaggccaag aattattctg gacgtttcac ctgctggtgg ctgacgacaa ttctaagatg cgaggccaag aattattctg gacgtttcac ctgctggtgg ctgacgacaa 720 720 tcagtactga tttgacatto agtgtcaaaa gcagcagagg ctcttctgac ccccaaggggg tcagtactga tttgacattc agtgtcaaaa gcagcagagg ctcttctgac ccccaagggg 780 780 tgacgtgcgg agctgctaca ctcagcgccg agagagtcag aggggacaac aaggagtatg tgacgtgcgg agctgctaca ctcagcgccg agagagtcag aggggacaac aaggagtatg 840 840

agtactcagt ggagtgccag gaggacagtg cctgcccagc tgctgaggag agtctgccca agtactcagt ggagtgccag gaggacagtg cctgcccagc tgctgaggag agtctgccca 900 900

ttgaggtcat ggtggatgcc gttcacaagc tcaagtatga aaactacacc agcagcttct ttgaggtcat ggtggatgcc gttcacaagc tcaagtatga aaactacacc agcagcttct 960 960 tcatcaggga catcatcaaa cctgacccac ccaagaactt gcagctgaag cccctgaaga tcatcaggga catcatcaaa cctgacccac ccaagaactt gcagctgaag cccctgaaga 1020 1020

acagcagaca ggtggaggtc agctgggagt accctgacac ctggagtact ccacattect acagcagaca ggtggaggtc agctgggagt accctgacac ctggagtact ccacattcct 1080 1080 acttctccct gacattctgc gttcaggtco agggcaagag caagagagaa aagaaagata acttctccct gacattctgc gttcaggtcc agggcaagag caagagagaa aagaaagata 1140 1140

gagtcttcac ggacaagaco tcagccacgg tcatctgccg caaaaatgcc agcattagcg gagtcttcac ggacaagacc tcagccacgg tcatctgccg caaaaatgcc agcattagcg 1200 1200 tgcgggccca ggaccgctad tatagctcat cttggagcga atgggcatct gtgccctgct tgcgggccca ggaccgctac tatagctcat cttggagcga atgggcatct gtgccctgct 1260 1260

ccggtggcgg tggcggcgga tctagaaacc tccccgtggc cactccagac ccaggaatgt ccggtggcgg tggcggcgga tctagaaacc tccccgtggc cactccagac ccaggaatgt 1320 1320

Page 106 Page 106

1X1 7S I09 50471‐706_601_SL.TXT tcccatgcct tcaccacagc cagaacctgc tgagggccgt cagcaacatg ctccagaagg 1380 08EI

ccagacaaac tctagaattt tacccttgca cttctgaaga gattgatcat gaagatatca 1440

caaaagataa aaccagcaca gtggaggcct gtttaccatt ggaattaacc aagaatgaga 1500 00ST

gttgcctaaa ttccagagag acctctttca taactaatgg gagttgcctg gcctccagaa 1560 09ST

agacctcttt tatgatggcc ctgtgcctta gtagtattta tgaagacttg aagatgtacc 1620 The aggtggagtt caagaccatg aatgcaaagc tgctgatgga ccccaagagg cagatctttc 1680 089T

tagatcaaaa catgctggca gttattgatg agctgatgca ggccctgaat ttcaacagtg 1740

agactgtgcc acaaaaatcc tcccttgaag aaccggattt ttataaaact aaaatcaagc 1800 008T

tctgcatact tcttcatgct ttcagaatca gagcagtgac tattgataga gtgatgagct 1860 098T

atctgaatgc ttccagagct aagaggggaa gcggagaggg cagaggaagt ctgctaacat 1920 The gcggtgacgt cgaggagaat cctggaccta ggctccctgc tcagctcctg gggctgctaa 1980 086T

tgctctgggt cccaggatcc agtgggcgca aagtgtgtaa cggaataggt attggtgaat 2040

ttaaagactc actctccata aatgctacga atattaaaca cttcaaaaac tgcacctcca 2100 00I2

tcagtggcga tctccacatc ctgccggtgg catttagggg tgactccttc acacatactc 2160

ctcctctgga tccacaggaa ctggatattc tgaaaaccgt aaaggaaatc acagggtttt 2220 0222

tgctgattca ggcttggcct gaaaacagga cggacctcca tgcctttgag aacctagaaa 2280 0822

tcatacgcgg caggaccaag caacatggtc agttttctct tgcagtcgtc agcctgaaca 2340 OTEL

e taacatcctt gggattacgc tccctcaagg agataagtga tggagatgtg ataatttcag 2400

gaaacaaaaa tttgtgctat gcaaatacaa taaactggaa aaaactgttt gggacctccg 2460

gtcagaaaac caaaattata agcaacagag gtgaaaacag ctgcaaggcc acaggccagg 2520 0252

tctgccatgc cttgtgctcc cccgagggct gctggggccc ggagcccagg gactgcgtct 2580 0852

ctggtggcgg tggctcgggc ggtggtgggt cgggtggcgg cggatctggt ggcggtggct 2640 797 cgttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg ctagtaacag 2700 00LZ

tggcctttat tattttctgg gtgaggagta agaggagcta aaacttgttt attgcagctt 2760 09/2

ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 2820 0282

997 9777887877 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgg 2873 EL82

Page 107 LOT aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<210> 137 <210> 137 <211> 3532 <211> 3532 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="FLC‐2B12/13‐2" <223> /note="FLC-2B12/13-2"

<400> 137 <400> 137 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60

tgctttatcg gggcggatca ctccgaaccc cgggaggtct atataagcag agctcgttta 180 tgctttatcg gggcggatca ctccgaaccc cgggaggtct atataagcag agctcgttta 180

gtgaaccctc attctggaga cggatcccga gccgagtgtt ttgacctcca tagaacagcc 240 gtgaaccctc attctggaga cggatcccga gccgagtgtt ttgacctcca tagaacagcc 240

gctaaatcca aggtaaggtc agaagagcta gcgccaccat ggattggacc tggattctgt 300 gctaaatcca aggtaaggtc agaagagcta gcgccaccat ggattggacc tggattctgt 300

ttctggtggc cgctgccaca agagtgcaca gcaactgggt gaatgtgatc agcgacctga 360 ttctggtggc cgctgccaca agagtgcaca gcaactgggt gaatgtgatc agcgacctga 360

agaagatcga ggatctgatc cagagcatgc acattgatgc caccctgtac acagaatctg 420 agaagatcga ggatctgatc cagagcatgo acattgatgo caccctgtac acagaatctg 420

atgtgcaccc tagctgtaaa gtgaccgcca tgaagtgttt tctgctggag ctgcaggtga 480 atgtgcaccc tagctgtaaa gtgaccgcca tgaagtgttt tctgctggag ctgcaggtga 480

tttctctgga aagcggagat gcctctatcc acgacacagt ggagaatctg atcatcctgg 540 tttctctgga aagcggagat gcctctatcc acgacacagt ggagaatctg atcatcctgg 540

ccaacaatag cctgagcagc aatggcaatg tgacagagtc tggctgtaag gagtgtgagg 600 ccaacaatag cctgagcagc aatggcaatg tgacagagtc tggctgtaag gagtgtgagg 600

agctggagga gaagaacatc aaggagtttc tgcagagctt tgtgcacatc gtgcagatgt 660 agctggagga gaagaacatc aaggagtttc tgcagagctt tgtgcacatc gtgcagatgt 660

tcatcaatac aagctctggc ggaggatctg gaggaggcgg atctggagga ggaggcagtg 720 tcatcaatac aagctctggc ggaggatctg gaggaggcgg atctggagga ggaggcagtg 720

gaggcggagg atctggcgga ggatctctgc agattacatg ccctcctcca atgtctgtgg 780 gaggcggagg atctggcgga ggatctctgc agattacatg ccctcctcca atgtctgtgg 780

agcacgccga tatttgggtg aagtcctaca gcctgtacag cagagagaga tacatctgca 840 agcacgccga tatttgggtg aagtcctaca gcctgtacag cagagagaga tacatctgca 840

acagcggctt taagagaaag gccggcacct cttctctgac agagtgcgtg ctgaataagg 900 acagcggctt taagagaaag gccggcacct cttctctgac agagtgcgtg ctgaataagg 900

ccacaaatgt ggcccactgg acaacaccta gcctgaagtg cattagagat cctgccctgg 960 ccacaaatgt ggcccactgg acaacaccta gcctgaagtg cattagagat cctgccctgg 960

tccaccagag gcctgcccct ccatctacag tgacaacagc cggagtgaca cctcagcctg 1020 tccaccagag gcctgcccct ccatctacag tgacaacagc cggagtgaca cctcagcctg 1020

aatctctgag cccttctgga aaagaacctg ccgccagctc tcctagctct aataataccg 1080 aatctctgag cccttctgga aaagaacctg ccgccagctc tcctagctct aataataccg 1080

Page 108 Page 108

50471‐706_601_SL.TXT 1x1'75 ccgccacaac agccgccatt gtgcctggat ctcagctgat gcctagcaag tctcctagca 1140

caggcacaac agagatcagc agccacgaat cttctcacgg aacaccttct cagaccaccg 1200 002T

ccaagaattg ggagctgaca gcctctgcct ctcaccagcc tccaggagtg tatcctcagg 1260 097T

gccactctga tacaacagtg gccatcagca catctacagt gctgctgtgt ggactgtctg 1320 OZET

ccgtgtctct gctggcctgt tacctgaagt ctagacagac acctcctctg gcctctgtgg 1380 08ET

agatggaggc catggaagcc ctgcctgtga catggggaac aagcagcaga gatgaggacc 1440

tggagaattg ttctcaccac ctgtgaaact tgtttattgc agcttataat ggttacaaat 1500 00ST

e aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 1560 9787788707 09ST

gtttgtccaa actcatcaat gtatcttatc atgtctggga gcgtgcgtga ggctccggtg 1620 0291

cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggagggggtc 1680 089T

ggcgattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg 1740

e tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc 1800 008T

gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac agcagccgct aaatccaagg 1860 098T

taaggtcaga agagctagcg ccaccatgaa gctgctgagc agcatcgagc aggcttgcga 1920 026T

catctgcagg ctgaagaagc tgaagtgcag caaggagaag cccaagtgcg ccaagtgcct 1980 Seededdeed 086T

gaagaacaac tgggagtgca gatacagccc caagaccaag aggagccccc tgaccagggc 2040 9702

See been ccacctgacc gaggtggaga gcaggctgga gaggctggag cagctgttcc tgctgatctt 2100 0012

ccccagggag gacctggaca tgatcctgaa gatggacagc ctgcaagaca tcaaggccct 2160 09TZ

gctgaccggc ctgttcgtgc aggacaacgt gaacaaggac gccgtgaccg acaggctggc 2220 0222

cagcgtggag accgacatgc ccctgaccct gaggcagcac aggatcagcg ccaccagcag 2280 0822

cagcgaggag agcagcaaca agggccagag gcagctgacc gtgagccccg agtttcccgg 2340

gatcaggccc gagtgcgtgg tgcccgagac ccagtgcgcc atgaaaagga aggagaagaa 2400

e ggcccagaag gagaaggaca agctgcccgt gagcaccacc accgtcgatg accacatgcc 2460

e ccccatcatg cagtgcgagc cccccccccc cgaggccgcc aggattcacg aggtcgtgcc 2520 0252

caggttcctg agcgacaagc tgctggtgac caacaggcag aagaacatcc cccagctgac 2580 0892

cgccaaccag cagttcctga tcgccaggct gatctggtat caggacggct acgagcagcc 2640

the Page 109 60T aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT cagcgacgag gacctgaaaa ggatcaccca gacctggcag caggccgacg acgagaacga 2700 cagcgacgag gacctgaaaa ggatcaccca gacctggcag caggccgacg acgagaacga 2700

ggagagcgac acccccttca ggcagatcac cgagatgacc atcctgaccg tgcagctgat 2760 ggagagcgac acccccttca ggcagatcad cgagatgacc atcctgaccg tgcagctgat 2760

cgtggagttc gccaagggcc tgcccggatt cgccaagatc agccagcccg accagatcac 2820 cgtggagttc gccaagggcc tgcccggatt cgccaagatc agccagcccg accagatcad 2820

cctgctgaag gcttgcagca gcgaggtgat gatgctgagg gtggccagga ggtacgacgc 2880 cctgctgaag gcttgcagca gcgaggtgat gatgctgagg gtggccagga ggtacgacgc 2880

cgccagcgac agcatcctgt tcgccaacaa ccaggcttac accagggaca actacaggaa 2940 cgccagcgac agcatcctgt tcgccaacaa ccaggcttac accagggaca actacaggaa 2940

ggctggcatg gccgaggtga tcgaggacct cctgcacttc tgcagatgta tgtacagcat 3000 ggctggcatg gccgaggtga tcgaggacct cctgcacttc tgcagatgta tgtacagcat 3000

ggccctggac aacatccact acgccctgct gaccgccgtg gtgatcttca gcgacaggcc 3060 ggccctggac aacatccact acgccctgct gaccgccgtg gtgatcttca gcgacaggcc 3060

cggcctggag cagccccagc tggtggagga gatccagagg tactacctga acaccctgag 3120 cggcctggag cagccccago tggtggagga gatccagagg tactacctga acaccctgag 3120

gatctacatc ctgaaccagc tgagcggcag cgccaggagc agcgtgatct acggcaagat 3180 gatctacatc ctgaaccago tgagcggcag cgccaggage agcgtgatct acggcaagat 3180

cctgagcatc ctgagcgagc tgaggaccct gggaatgcag aacagcaata tgtgtatcag 3240 cctgagcatc ctgagcgago tgaggaccct gggaatgcag aacagcaata tgtgtatcag 3240

cctgaagctg aagaacagga agctgccccc cttcctggag gagatttggg acgtggccga 3300 cctgaagctg aagaacagga agctgccccc cttcctggag gagatttggg acgtggccga 3300

catgagccac acccagcccc cccccatcct ggagagcccc accaacctgt gatgatgggt 3360 catgagccac acccagcccc cccccatcct ggagagcccc accaacctgt gatgatgggt 3360

ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc 3420 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc 3420

ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct 3480 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct 3480

ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aa 3532 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aa 3532

<210> 138 <210> 138 <211> 4423 <211> 4423 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="XON‐02" <223> /note="XON-02"

<400> 138 <400> 138 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60

Page 110 Page 110

50471‐706_601_SL.TXT 7S gtgaaccctc attctggaga cggatcccga gccgagtgtt ttgacctcca tagaacagcc 240

EXP the gctaaatcca aggtaaggtc agaagagcta gcgccaccat ggattggacc tggattctgt 300 00E

ttctggtggc cgctgccaca agagtgcaca gcaactgggt gaatgtgatc agcgacctga 360 09E

agaagatcga ggatctgatc cagagcatgc acattgatgc caccctgtac acagaatctg 420

atgtgcaccc tagctgtaaa gtgaccgcca tgaagtgttt tctgctggag ctgcaggtga 480 08/

tttctctgga aagcggagat gcctctatcc acgacacagt ggagaatctg atcatcctgg 540

ccaacaatag cctgagcagc aatggcaatg tgacagagtc tggctgtaag gagtgtgagg 600 009

the agctggagga gaagaacatc aaggagtttc tgcagagctt tgtgcacatc gtgcagatgt 660 099

tcatcaatac aagctctggc ggaggatctg gaggaggcgg atctggagga ggaggcagtg 720 OZL

gaggcggagg atctggcgga ggatctctgc agattacatg ccctcctcca atgtctgtgg 780 08L

agcacgccga tatttgggtg aagtcctaca gcctgtacag cagagagaga tacatctgca 840

acagcggctt taagagaaag gccggcacct cttctctgac agagtgcgtg ctgaataagg 900 006

ccacaaatgt ggcccactgg acaacaccta gcctgaagtg cattagagat cctgccctgg 960 096

tccaccagag gcctgcccct ccatctacag tgacaacagc cggagtgaca cctcagcctg 1020 0201

aatctctgag cccttctgga aaagaacctg ccgccagctc tcctagctct aataataccg 1080 080I

ccgccacaac agccgccatt gtgcctggat ctcagctgat gcctagcaag tctcctagca 1140

caggcacaac agagatcagc agccacgaat cttctcacgg aacaccttct cagaccaccg 1200

ccaagaattg ggagctgaca gcctctgcct ctcaccagcc tccaggagtg tatcctcagg 1260 The gccactctga tacaacagtg gccatcagca catctacagt gctgctgtgt ggactgtctg 1320 OZET

ccgtgtctct gctggcctgt tacctgaagt ctagacagac acctcctctg gcctctgtgg 1380 08EI

agatggaggc catggaagcc ctgcctgtga catggggaac aagcagcaga gatgaggacc 1440

tggagaattg ttctcaccac ctgtgaaact tgtttattgc agcttataat ggttacaaat 1500 00ST

aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 1560 09ST

gtttgtccaa actcatcaat gtatcttatc atgtctggga gcgtgcgtga ggctccggtg 1620 The cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggagggggtc 1680 089T

ggcgattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg 1740

Page 111 III aged

50471‐706_601_SL.TXT 1x1'7S tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc 1800 008T

gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac agcagccgct aaatccaagg 1860 098T

taaggtcaga agagctagcg ccaccatgct gctgctggtg accagcctgc tgctgtgtga 1920 026T

gctgccccac cccgcctttc tgctgatccc cgacatccag atgacccaga ccacctccag 1980 086T

cctgagcgcc agcctgggcg accgggtgac catcagctgc cgggccagcc aggacatcag 2040 9707

caagtacctg aactggtatc agcagaagcc cgacggcacc gtcaagctgc tgatctacca 2100 0012

caccagccgg ctgcacagcg gcgtgcccag ccggtttagc ggcagcggct ccggcaccga 2160 09T2

ctacagcctg accatctcca acctggagca ggaggacatc gccacctact tttgccagca 2220 0222

gggcaacaca ctgccctaca cctttggcgg cggaacaaag ctggagatca ccggcagcac 2280 0822

ctccggcagc ggcaagcctg gcagcggcga gggcagcacc aagggcgagg tgaagctgca 2340

ggagagcggc cctggcctgg tggcccccag ccagagcctg agcgtgacct gtaccgtgtc 2400

cggcgtgtcc ctgcccgact acggcgtgtc ctggatccgg cagcccccta ggaagggcct 2460

ggagtggctg ggcgtgatct ggggcagcga gaccacctac tacaacagcg ccctgaagag 2520 0252

ccggctgacc atcatcaagg acaacagcaa gagccaggtg ttcctgaaga tgaacagcct 2580 0857

gcagaccgac gacaccgcca tctactactg tgccaagcac tactactacg gcggcagcta 2640

cgccatggac tactggggcc agggcaccag cgtgaccgtg tccagcaagc ccaccaccac 2700 00LZ

ccctgcccct agacctccaa ccccagcccc tacaatcgcc agccagcccc tgagcctgag 2760 09/2

gcccgaagcc tgtagacctg ccgctggcgg agccgtgcac accagaggcc tggatttcgc 2820 0807779887 0787

ctgcgacatc tacatctggg cccctctggc cggcacctgt ggcgtgctgc tgctgagcct 2880 0879878588 0887

ggtcatcacc ctgtactgca accaccggaa taggagcaag cggagcagag gcggccacag 2940 9767

cgactacatg aacatgaccc cccggaggcc tggccccacc cggaagcact accagcccta 3000 000E

cgcccctccc agggacttcg ccgcctaccg gagccgggtg aagttcagcc ggagcgccga 3060 090E

cgcccctgcc taccagcagg gccagaacca gctgtacaac gagctgaacc tgggccggag 3120 OZIE

ggaggagtac gacgtgctgg acaagcggag aggccgggac cctgagatgg gcggcaagcc 3180 08TE

ee ccggagaaag aaccctcagg agggcctgta taacgaactg cagaaagaca agatggccga 3240 e Street ggcctacagc gagatcggca tgaagggcga gcggcggagg ggcaagggcc acgacggcct 3300 00EE

es: Page 112 ZII aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT gtaccagggc ctgagcaccg ccaccaagga tacctacgac gccctgcaca tgcaggccct 3360 gtaccagggc ctgagcaccg ccaccaagga tacctacgac gccctgcaca tgcaggccct 3360

gccccccaga agggccaaga ggagtggcag cggcgagggc agaggaagtc ttctaacatg 3420 gcccccccaga agggccaaga ggagtggcag cggcgagggo agaggaagto ttctaacatg 3420

cggtgacgtg gaggagaatc ccggccctat gaggctccct gctcagctcc tggggctgct 3480 cggtgacgtg gaggagaato ccggccctat gaggctccct gctcagctcc tggggctgct 3480

aatgctctgg gtcccaggat ccagtgggcg caaagtgtgt aacggaatag gtattggtga 3540 aatgctctgg gtcccaggat ccagtgggcg caaagtgtgt aacggaatag gtattggtga 3540

atttaaagac tcactctcca taaatgctac gaatattaaa cacttcaaaa actgcacctc 3600 atttaaagac tcactctcca taaatgctac gaatattaaa cacttcaaaa actgcacctc 3600

catcagtggc gatctccaca tcctgccggt ggcatttagg ggtgactcct tcacacatac 3660 catcagtggc gatctccaca tcctgccggt ggcatttagg ggtgactcct tcacacatad 3660

tcctcctctg gatccacagg aactggatat tctgaaaacc gtaaaggaaa tcacagggtt 3720 tcctcctctg gatccacagg aactggatat tctgaaaacc gtaaaggaaa tcacagggtt 3720

tttgctgatt caggcttggc ctgaaaacag gacggacctc catgcctttg agaacctaga 3780 tttgctgatt caggcttggc ctgaaaacag gacggaccto catgcctttg agaacctaga 3780

aatcatacgc ggcaggacca agcaacatgg tcagttttct cttgcagtcg tcagcctgaa 3840 aatcatacgc ggcaggacca agcaacatgg tcagttttct cttgcagtcg tcagcctgaa 3840

cataacatcc ttgggattac gctccctcaa ggagataagt gatggagatg tgataatttc 3900 cataacatcc ttgggattac gctccctcaa ggagataagt gatggagatg tgataattto 3900

aggaaacaaa aatttgtgct atgcaaatac aataaactgg aaaaaactgt ttgggacctc 3960 aggaaacaaa aatttgtgct atgcaaatac aataaactgg aaaaaactgt ttgggacctc 3960

cggtcagaaa accaaaatta taagcaacag aggtgaaaac agctgcaagg ccacaggcca 4020 cggtcagaaa accaaaatta taagcaacag aggtgaaaac agctgcaagg ccacaggcca 4020

ggtctgccat gccttgtgct cccccgaggg ctgctggggc ccggagccca gggactgcgt 4080 ggtctgccat gccttgtgct cccccgaggg ctgctggggc ccggagccca gggactgcgt 4080

ctctggtggc ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg gtggcggtgg 4140 ctctggtggc ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg gtggcggtgg 4140

ctcgttttgg gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac 4200 ctcgttttgg gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac 4200

agtggccttt attattttct gggtgaggag taagaggagc taatgatggg tggcatccct 4260 agtggccttt attattttct gggtgaggag taagaggago taatgatggg tggcatccct 4260

gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc 4320 gtgacccctc cccagtgcct ctcctggccc tggaagttgo cactccagtg cccaccagco 4320

ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta 4380 ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta 4380

tggggtggag gggggtggta tggagcaagg ggcaagttgg gaa 4423 tggggtggag gggggtggta tggagcaagg ggcaagttgg gaa 4423

<210> 139 <210> 139 <211> 4423 <211> 4423 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= Description of Artificial Sequence: Synthetic polynucleotide" polynucleotide"

<220> <220> <221> source <221> source <223> /note="XON‐02(h)" <223> /note="XON-02(h)' "

Page 113 Page 113

50471‐706_601_SL.TXT

<400> 139 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60 09

gagaagttgg ggggaggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt 120 99999.9999 OZI

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 08T

gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240

acagcagccg ctaaatccaa ggtaaggtca gaagagctag cgccaccatg ctgctgctgg 300 00E

tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatcc 360 09E

agatgaccca gaccacctcc agcctgagcg ccagcctggg cgaccgggtg accatcagct 420

gccgggccag ccaggacatc agcaagtacc tgaactggta tcagcagaag cccgacggca 480 08/7

ccgtcaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc agccggttta 540

gcggcagcgg ctccggcacc gactacagcc tgaccatctc caacctggag caggaggaca 600 009

e tcgccaccta cttttgccag cagggcaaca cactgcccta cacctttggc ggcggaacaa 660 099

agctggagat caccggcagc acctccggca gcggcaagcc tggcagcggc gagggcagca 720 OZL

ccaagggcga ggtgaagctg caggagagcg gccctggcct ggtggccccc agccagagcc 780 08L

tgagcgtgac ctgtaccgtg tccggcgtgt ccctgcccga ctacggcgtg tcctggatcc 840 2000 ggcagccccc taggaagggc ctggagtggc tgggcgtgat ctggggcagc gagaccacct 900 006

actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacagc aagagccagg 960 096

tgttcctgaa gatgaacagc ctgcagaccg acgacaccgc catctactac tgtgccaagc 1020

the actactacta cggcggcagc tacgccatgg actactgggg ccagggcacc agcgtgaccg 1080 080T

tgtccagcaa gcccaccacc acccctgccc ctagacctcc aaccccagcc cctacaatcg 1140

ccagccagcc cctgagcctg aggcccgaag cctgtagacc tgccgctggc ggagccgtgc 1200 Seed acaccagagg cctggatttc gcctgcgaca tctacatctg ggcccctctg gccggcacct 1260 092T

gtggcgtgct gctgctgagc ctggtcatca ccctgtactg caaccaccgg aataggagca 1320 OZET

agcggagcag aggcggccac agcgactaca tgaacatgac cccccggagg cctggcccca 1380 08ET

cccggaagca ctaccagccc tacgcccctc ccagggactt cgccgcctac cggagccggg 1440

tgaagttcag ccggagcgcc gacgcccctg cctaccagca gggccagaac cagctgtaca 1500 00ST been

e Page 114 aged

50471‐706_601_SL.TXT 1X1 TS I09 90 acgagctgaa cctgggccgg agggaggagt acgacgtgct ggacaagcgg agaggccggg 1560 09ST

accctgagat gggcggcaag ccccggagaa agaaccctca ggagggcctg tataacgaac 1620 The tgcagaaaga caagatggcc gaggcctaca gcgagatcgg catgaagggc gagcggcgga 1680 089T

ggggcaaggg ccacgacggc ctgtaccagg gcctgagcac cgccaccaag gatacctacg 1740

acgccctgca catgcaggcc ctgcccccca gaagggccaa gaggagtggc agcggcgagg 1800 008T

gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct atgaggctcc 1860 098T

ctgctcagct cctggggctg ctaatgctct gggtcccagg atccagtggg cgcaaagtgt 1920 026T

gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct acgaatatta 1980 086T

aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg gtggcattta 2040

ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat attctgaaaa 2100 00I2

the ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac aggacggacc 2160 09T2

tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat ggtcagtttt 2220 0222

ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc aaggagataa 2280 0822

gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat acaataaact 2340 OTES

ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac agaggtgaaa 2400

acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag ggctgctggg 2460

gcccggagcc cagggactgc gtctctggtg gcggtggctc gggcggtggt gggtcgggtg 2520 0252

gcggcggatc tggtggcggt ggctcgtttt gggtgctggt ggtggttggt ggagtcctgg 2580 1997788.89 0852

cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg agtaagagga 2640

gctaatgatg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt 2700 00/2

credit 997999999e e gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac 2760

the 09/2

taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt 2820 0282

gggaaattgt tcggagcagt gcggcgcgtt tagcggagta ctgtcctccg atattaatcg 2880 0887

gggcagacta ttccggggtt taccggcgca ctctcgcccg aacttcaccg gcggtctttc 2940 797 gtccgtgctt tatcggggcg gatcactccg aaccccggga ggtctatata agcagagctc 3000 000E

gtttagtgaa ccctcattct ggagacggat cccgagccga gtgttttgac ctccatagaa 3060 090E

e Page 115 STT aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT cagccgctaa atccaaggta aggtcagaag agctagcgcc accatggatt ggacctggat cagccgctaa atccaaggta aggtcagaag agctagcgcc accatggatt ggacctggat 3120 3120 tctgtttctg gtggccgctg ccacaagagt gcacagcaac tgggtgaatg tgatcagcga tctgtttctg gtggccgctg ccacaagagt gcacagcaac tgggtgaatg tgatcagcga 3180 3180 cctgaagaag atcgaggatc tgatccagag catgcacatt gatgccaccc tgtacacaga cctgaagaag atcgaggatc tgatccagag catgcacatt gatgccaccc tgtacacaga 3240 3240 atctgatgtg caccctagct gtaaagtgac cgccatgaag tgttttctgc tggagctgca atctgatgtg caccctagct gtaaagtgac cgccatgaag tgttttctgc tggagctgca 3300 3300 ggtgatttct ctggaaagcg gagatgcctc tatccacgac acagtggaga atctgatcat ggtgatttct ctggaaagcg gagatgcctc tatccacgac acagtggaga atctgatcat 3360 3360 cctggccaac aatagcctga gcagcaatgg caatgtgaca gagtctggct gtaaggagtg cctggccaac aatagcctga gcagcaatgg caatgtgaca gagtctggct gtaaggagtg 3420 3420 tgaggagctg gaggagaaga acatcaagga gtttctgcag agctttgtgc acatcgtgca tgaggagctg gaggagaaga acatcaagga gtttctgcag agctttgtgc acatcgtgca 3480 3480 gatgttcatc aatacaagct ctggcggagg atctggagga ggcggatctg gaggaggagg gatgttcatc aatacaagct ctggcggagg atctggagga ggcggatctg gaggaggagg 3540 3540 cagtggaggc ggaggatctg gcggaggatc tctgcagatt acatgccctc ctccaatgtc cagtggaggc ggaggatctg gcggaggatc tctgcagatt acatgccctc ctccaatgtc 3600 3600 tgtggagcac gccgatattt gggtgaagtc ctacagcctg tacagcagag agagatacat tgtggagcac gccgatattt gggtgaagtc ctacagcctg tacagcagag agagatacat 3660 3660 ctgcaacagc ggctttaaga gaaaggccgg cacctcttct ctgacagagt gcgtgctgaa ctgcaacagc ggctttaaga gaaaggccgg cacctcttct ctgacagagt gcgtgctgaa 3720 3720 taaggccaca aatgtggccc actggacaac acctagcctg aagtgcatta gagatcctgc taaggccaca aatgtggccc actggacaac acctagcctg aagtgcatta gagatcctgc 3780 3780 cctggtccac cagaggcctg cccctccatc tacagtgaca acagccggag tgacacctca cctggtccac cagaggcctg cccctccatc tacagtgaca acagccggag tgacacctca 3840 3840 gcctgaatct ctgagccctt ctggaaaaga acctgccgcc agctctccta gctctaataa gcctgaatct ctgagccctt ctggaaaaga acctgccgcc agctctccta gctctaataa 3900 3900 taccgccgcc acaacagccg ccattgtgcc tggatctcag ctgatgccta gcaagtctcc taccgccgcc acaacagccg ccattgtgcc tggatctcag ctgatgccta gcaagtctcc 3960 3960 tagcacaggc acaacagaga tcagcagcca cgaatcttct cacggaacac cttctcagac tagcacaggc acaacagaga tcagcagcca cgaatcttct cacggaacac cttctcagac 4020 4020 caccgccaag aattgggago tgacagcctc tgcctctcac cagcctccag gagtgtatcc caccgccaag aattgggagc tgacagcctc tgcctctcac cagcctccag gagtgtatcc 4080 4080 tcagggccac tctgatacaa cagtggccat cagcacatct acagtgctgc tgtgtggact tcagggccac tctgatacaa cagtggccat cagcacatct acagtgctgc tgtgtggact 4140 4140 gtctgccgtg tctctgctgg cctgttacct gaagtctaga cagacacctc ctctggcctc gtctgccgtg tctctgctgg cctgttacct gaagtctaga cagacacctc ctctggcctc 4200 4200 tgtggagatg gaggccatgg aagccctgcc tgtgacatgg ggaacaagca gcagagatga tgtggagatg gaggccatgg aagccctgcc tgtgacatgg ggaacaagca gcagagatga 4260 4260 ggacctggag aattgttctc accacctgtg aaacttgttt attgcagctt ataatggtta ggacctggag aattgttctc accacctgtg aaacttgttt attgcagctt ataatggtta 4320 4320 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 4380 4380 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgg ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgg 4423 4423

<210> 140 <210> 140 <211> 4487 <211> 4487 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

Page 116 Page 116

<220> <220> <221> source <221> source <223> /note="FLC‐2B8‐1" <223> /note="FLC-2B8-1"

<400> 140 <400> 140 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagago gcacatcgcc cacagtcccc 60

tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatcc 360 tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatco 360

agatgaccca gaccacctcc agcctgagcg ccagcctggg cgaccgggtg accatcagct 420 agatgaccca gaccacctcc agcctgagcg ccagcctggg cgaccgggtg accatcagct 420

gccgggccag ccaggacatc agcaagtacc tgaactggta tcagcagaag cccgacggca 480 gccgggccag ccaggacato agcaagtacc tgaactggta tcagcagaag cccgacggca 480

ccgtcaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc agccggttta 540 ccgtcaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc agccggttta 540

gcggcagcgg ctccggcacc gactacagcc tgaccatctc caacctggag caggaggaca 600 gcggcagcgg ctccggcacc gactacagcc tgaccatctc caacctggag caggaggaca 600

tcgccaccta cttttgccag cagggcaaca cactgcccta cacctttggc ggcggaacaa 660 tcgccaccta cttttgccag cagggcaaca cactgcccta cacctttggc ggcggaacaa 660

agctggagat caccggcagc acctccggca gcggcaagcc tggcagcggc gagggcagca 720 agctggagat caccggcage acctccggca gcggcaagcc tggcagcggc gagggcagca 720

ccaagggcga ggtgaagctg caggagagcg gccctggcct ggtggccccc agccagagcc 780 ccaagggcga ggtgaagctg caggagagcg gccctggcct ggtggccccc agccagagcc 780

tgagcgtgac ctgtaccgtg tccggcgtgt ccctgcccga ctacggcgtg tcctggatcc 840 tgagcgtgac ctgtaccgtg tccggcgtgt ccctgcccga ctacggcgtg tcctggatcc 840

ggcagccccc taggaagggc ctggagtggc tgggcgtgat ctggggcagc gagaccacct 900 ggcagccccc taggaagggc ctggagtggc tgggcgtgat ctggggcagc gagaccacct 900

actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacagc aagagccagg 960 actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacago aagagccagg 960

tgttcctgaa gatgaacagc ctgcagaccg acgacaccgc catctactac tgtgccaagc 1020 tgttcctgaa gatgaacagc ctgcagaccg acgacaccgc catctactad tgtgccaagc 1020

actactacta cggcggcagc tacgccatgg actactgggg ccagggcacc agcgtgaccg 1080 actactacta cggcggcagc tacgccatgg actactgggg ccagggcaco agcgtgaccg 1080

tgtccagcaa gcccaccacc acccctgccc ctagacctcc aaccccagcc cctacaatcg 1140 tgtccagcaa gcccaccacc acccctgccc ctagacctcc aaccccagcc cctacaatcg 1140

ccagccagcc cctgagcctg aggcccgaag cctgtagacc tgccgctggc ggagccgtgc 1200 ccagccagcc cctgagcctg aggcccgaag cctgtagacc tgccgctggc ggagccgtgc 1200

acaccagagg cctggatttc gcctgcgaca tctacatctg ggcccctctg gccggcacct 1260 acaccagagg cctggattto gcctgcgaca tctacatctg ggcccctctg gccggcacct 1260

Page 117 Page 117

50471‐706_601_SL.TXT gtggcgtgct gctgctgagc ctggtcatca ccctgtactg caaccaccgg aataggagca 1320 OZET

agcggagcag aggcggccac agcgactaca tgaacatgac cccccggagg cctggcccca 1380 08ET

cccggaagca ctaccagccc tacgcccctc ccagggactt cgccgcctac cggagccggg 1440

tgaagttcag ccggagcgcc gacgcccctg cctaccagca gggccagaac cagctgtaca 1500 00ST

acgagctgaa cctgggccgg agggaggagt acgacgtgct ggacaagcgg agaggccggg 1560 09ST

989 the accctgagat gggcggcaag ccccggagaa agaaccctca ggagggcctg tataacgaac 1620 079T

tgcagaaaga caagatggcc gaggcctaca gcgagatcgg catgaagggc gagcggcgga 1680 089T

ggggcaaggg ccacgacggc ctgtaccagg gcctgagcac cgccaccaag gatacctacg 1740

acgccctgca catgcaggcc ctgcccccca gaagagctaa gaggggaagc ggagagggca 1800 008T

e. ee gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggacctggc cccaagaaga 1860 098T

aaaggaaggt ggcccccccc accgacgtga gcctgggcga cgagctgcac ctggacggcg 1920 789ee99eee 026T

aggacgtggc catggcccac gccgacgccc tggacgactt cgacctggac atgctgggcg 1980 086T

acggcgacag ccccggcccc ggcttcaccc cccacgacag cgccccctac ggcgccctgg 2040

acatggccga cttcgagttc gagcagatgt tcaccgacgc cctgggcatc gacgagtacg 2100 0012

gcggcgaatt cgagatgccc gtggacagga ttctggaggc cgaactcgcc gtggagcaga 2160 09T2

aaagcgacca gggcgtggag ggccccggcg gaaccggcgg cagcggcagc agccccaacg 2220 0222

e accccgtgac caacatctgc caggccgccg acaagcagct gttcaccctg gtggagtggg 2280 0822

ccaagaggat tccccacttc agcagcctgc ccctggacga ccaggtgatc ctgctgaggg 2340 OTEC

ccggatggaa cgagctgctg atcgccagct tcagccacag gagcatcgac gtgagggacg 2400

the e gcatcctgct ggccaccggc ctgcacgtcc ataggaacag cgcccacagc gccggagtgg 2460

gcgccatctt cgacagggtg ctgaccgagc tggtgagcaa gatgagggac atgaggatgg 2520 0252

acaagaccga gctgggctgc ctgagggcca tcatcctgtt caaccccgag gtgaggggcc 2580 0852

tgaaaagcgc ccaggaggtg gagctgctga gggagaaggt gtacgccgcc ctggaggagt 2640 788ee9e999

e acaccaggac cacccacccc gacgagcccg gcagattcgc caagctgctg ctgaggctgc 2700

Page 118 8TT aged 00LZ

ccagcctgag gagcatcggc ctgaagtgcc tggagcacct gttcttcttc aggctgatcg 2760 09/2

gcgacgtgcc catcgacacc ttcctgatgg agatgctgga gagccccagc gacagcagag 2820

50471‐706_601_SL.TXT 1X1 is ctaagagggg aagcggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga 2880 0887

atcctggacc taagctgctg agcagcatcg agcaggcttg cgacatctgc aggctgaaga 2940

agctgaagtg cagcaaggag aagcccaagt gcgccaagtg cctgaagaac aactgggagt 3000 000E

gcagatacag ccccaagacc aagaggagcc ccctgaccag ggcccacctg accgaggtgg 3060 090E

agagcaggct ggagaggctg gagcagctgt tcctgctgat cttccccagg gaggacctgg 3120 OZIE

acatgatcct gaagatggac agcctgcaag acatcaaggc cctgctgacc ggcctgttcg 3180 08TE

tgcaggacaa cgtgaacaag gacgccgtga ccgacaggct ggccagcgtg gagaccgaca 3240

tgcccctgac cctgaggcag cacaggatca gcgccaccag cagcagcgag gagagcagca 3300 00EE

acaagggcca gaggcagctg accgtgagcc ccgagtttcc cgggatcagg cccgagtgcg 3360 09EE

tggtgcccga gacccagtgc gccatgaaaa ggaaggagaa gaaggcccag aaggagaagg 3420 89eededdee

acaagctgcc cgtgagcacc accaccgtcg atgaccacat gccccccatc atgcagtgcg 3480

agcccccccc ccccgaggcc gccaggattc acgaggtcgt gcccaggttc ctgagcgaca 3540

agctgctggt gaccaacagg cagaagaaca tcccccagct gaccgccaac cagcagttcc 3600 009E

tgatcgccag gctgatctgg tatcaggacg gctacgagca gcccagcgac gaggacctga 3660 099 aaaggatcac ccagacctgg cagcaggccg acgacgagaa cgaggagagc gacaccccct 3720 OZLE

tcaggcagat caccgagatg accatcctga ccgtgcagct gatcgtggag ttcgccaagg 3780 08LE

gcctgcccgg attcgccaag atcagccagc ccgaccagat caccctgctg aaggcttgca 3840

gcagcgaggt gatgatgctg agggtggcca ggaggtacga cgccgccagc gacagcatcc 3900 006E

0800080080 e tgttcgccaa caaccaggct tacaccaggg acaactacag gaaggctggc atggccgagg 3960 0968

tgatcgagga cctcctgcac ttctgcagat gtatgtacag catggccctg gacaacatcc 4020 0201

actacgccct gctgaccgcc gtggtgatct tcagcgacag gcccggcctg gagcagcccc 4080 080/7

agctggtgga ggagatccag aggtactacc tgaacaccct gaggatctac atcctgaacc 4140

agctgagcgg cagcgccagg agcagcgtga tctacggcaa gatcctgagc atcctgagcg 4200

agctgaggac cctgggaatg cagaacagca atatgtgtat cagcctgaag ctgaagaaca 4260 the ggaagctgcc ccccttcctg gaggagattt gggacgtggc cgacatgagc cacacccagc 4320

been e Page 119 6TT aged checked ccccccccat cctggagagc cccaccaacc tgtgaaactt gtttattgca gcttataatg 4380

50471‐706_601_SL.TXT 50471-706_601_SL.TXT gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 4440 gttacaaata aagcaatage atcacaaatt tcacaaataa agcatttttt tcactgcatt 4440

ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgg 4487 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgg 4487

<210> 141 <210> 141 <211> 4509 <211> 4509 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="FLC‐2A3‐1" <223> /note="FLC-2A3-1"

<400> 141 <400> 141 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60

ggcagccccc taggaagggc ctggagtggc tgggcgtgat ctggggcagc gagaccacct 900 ggcagccccc taggaagggc ctggagtggc tgggcgtgat ctggggcago gagaccacct 900

actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacagc aagagccagg 960 actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacage aagagccagg 960

Page 120 Page 120

50471‐706_601_SL.TXT 1X1'7S tgttcctgaa gatgaacagc ctgcagaccg acgacaccgc catctactac tgtgccaagc 1020 020T

actactacta cggcggcagc tacgccatgg actactgggg ccagggcacc agcgtgaccg 1080 080T

tgtccagcaa gcccaccacc acccctgccc ctagacctcc aaccccagcc cctacaatcg 1140

ccagccagcc cctgagcctg aggcccgaag cctgtagacc tgccgctggc ggagccgtgc 1200

acaccagagg cctggatttc gcctgcgaca tctacatctg ggcccctctg gccggcacct 1260 092T

gtggcgtgct gctgctgagc ctggtcatca ccctgtactg caaccaccgg aataggagca 1320 OZET

agcggagcag aggcggccac agcgactaca tgaacatgac cccccggagg cctggcccca 1380 08EI

cccggaagca ctaccagccc tacgcccctc ccagggactt cgccgcctac cggagccggg 1440

tgaagttcag ccggagcgcc gacgcccctg cctaccagca gggccagaac cagctgtaca 1500 00ST

acgagctgaa cctgggccgg agggaggagt acgacgtgct ggacaagcgg agaggccggg 1560 09ST

the accctgagat gggcggcaag ccccggagaa agaaccctca ggagggcctg tataacgaac 1620 029T been tgcagaaaga caagatggcc gaggcctaca gcgagatcgg catgaagggc gagcggcgga 1680 089T

ggggcaaggg ccacgacggc ctgtaccagg gcctgagcac cgccaccaag gatacctacg 1740

acgccctgca catgcaggcc ctgcccccca gaagagctaa gaggggaagc ggagagggca 1800 008T

gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggacctggc cccaagaaga 1860 098T

799ee99eee eee aaaggaaggt ggcccccccc accgacgtga gcctgggcga cgagctgcac ctggacggcg 1920 026T

aggacgtggc catggcccac gccgacgccc tggacgactt cgacctggac atgctgggcg 1980 086T

acggcgacag ccccggcccc ggcttcaccc cccacgacag cgccccctac ggcgccctgg 2040

acatggccga cttcgagttc gagcagatgt tcaccgacgc cctgggcatc gacgagtacg 2100 0012

gcggcgaatt cgagatgccc gtggacagga ttctggaggc cgaactcgcc gtggagcaga 2160 09T2

aaagcgacca gggcgtggag ggccccggcg gaaccggcgg cagcggcagc agccccaacg 2220 0222

e accccgtgac caacatctgc caggccgccg acaagcagct gttcaccctg gtggagtggg 2280 0822

ccaagaggat tccccacttc agcagcctgc ccctggacga ccaggtgatc ctgctgaggg 2340

2000 e ccggatggaa cgagctgctg atcgccagct tcagccacag gagcatcgac gtgagggacg 2400

gcatcctgct ggccaccggc ctgcacgtcc ataggaacag cgcccacagc gccggagtgg 2460

gcgccatctt cgacagggtg ctgaccgagc tggtgagcaa gatgagggac atgaggatgg 2520 0252

Page 121 TEL and

50471‐706_601_SL.TXT acaagaccga gctgggctgc ctgagggcca tcatcctgtt caaccccgag gtgaggggcc 2580 0857

tgaaaagcgc ccaggaggtg gagctgctga gggagaaggt gtacgccgcc ctggaggagt 2640

e acaccaggac cacccacccc gacgagcccg gcagattcgc caagctgctg ctgaggctgc 2700 00LZ

ccagcctgag gagcatcggc ctgaagtgcc tggagcacct gttcttcttc aggctgatcg 2760 09/2

gcgacgtgcc catcgacacc ttcctgatgg agatgctgga gagccccagc gacagctgag 2820 0787

catgcactag ttttataatt tcttcttcca gaatttctga cattttataa tttcttcttc 2880 0887

cagaagactc acaacctcca tatggccacc atgaagctgc tgagcagcat cgagcaggct 2940 9767

tgcgacatct gcaggctgaa gaagctgaag tgcagcaagg agaagcccaa gtgcgccaag 3000 000E

ee tgcctgaaga acaactggga gtgcagatac agccccaaga ccaagaggag ccccctgacc 3060 090E

agggcccacc tgaccgaggt ggagagcagg ctggagaggc tggagcagct gttcctgctg 3120 OZIE

88e e e atcttcccca gggaggacct ggacatgatc ctgaagatgg acagcctgca agacatcaag 3180 08TE

gccctgctga ccggcctgtt cgtgcaggac aacgtgaaca aggacgccgt gaccgacagg 3240

ctggccagcg tggagaccga catgcccctg accctgaggc agcacaggat cagcgccacc 3300 00EE

agcagcagcg aggagagcag caacaagggc cagaggcagc tgaccgtgag ccccgagttt 3360 0988

cccgggatca ggcccgagtg cgtggtgccc gagacccagt gcgccatgaa aaggaaggag 3420

aagaaggccc agaaggagaa ggacaagctg cccgtgagca ccaccaccgt cgatgaccac 3480 2000 atgcccccca tcatgcagtg cgagcccccc ccccccgagg ccgccaggat tcacgaggtc 3540

gtgcccaggt tcctgagcga caagctgctg gtgaccaaca ggcagaagaa catcccccag 3600 0098

ctgaccgcca accagcagtt cctgatcgcc aggctgatct ggtatcagga cggctacgag 3660 099 The cagcccagcg acgaggacct gaaaaggatc acccagacct ggcagcaggc cgacgacgag 3720 OZLE

aacgaggaga gcgacacccc cttcaggcag atcaccgaga tgaccatcct gaccgtgcag 3780 08LE

ede ctgatcgtgg agttcgccaa gggcctgccc ggattcgcca agatcagcca gcccgaccag 3840

atcaccctgc tgaaggcttg cagcagcgag gtgatgatgc tgagggtggc caggaggtac 3900 006E

gacgccgcca gcgacagcat cctgttcgcc aacaaccagg cttacaccag ggacaactac 3960 0968

aggaaggctg gcatggccga ggtgatcgag gacctcctgc acttctgcag atgtatgtac 4020 0701

agcatggccc tggacaacat ccactacgcc ctgctgaccg ccgtggtgat cttcagcgac 4080 080t

Page 122 ZZI aged

50471‐706_601_SL.TXT 50471-706_601_SL.TX aggcccggcc tggagcagcc ccagctggtg gaggagatcc agaggtacta cctgaacacc 4140 aggcccggcc tggagcagcc ccagctggtg gaggagatco agaggtacta cctgaacacc 4140

ctgaggatct acatcctgaa ccagctgagc ggcagcgcca ggagcagcgt gatctacggc 4200 ctgaggatct acatcctgaa ccagctgage ggcagcgcca ggagcagcgt gatctacggo 4200

aagatcctga gcatcctgag cgagctgagg accctgggaa tgcagaacag caatatgtgt 4260 aagatcctga gcatcctgag cgagctgagg accctgggaa tgcagaacag caatatgtgt 4260

atcagcctga agctgaagaa caggaagctg ccccccttcc tggaggagat ttgggacgtg 4320 atcagcctga agctgaagaa caggaagctg ccccccttcc tggaggagat ttgggacgtg 4320

gccgacatga gccacaccca gccccccccc atcctggaga gccccaccaa cctgtgaaac 4380 gccgacatga gccacaccca gccccccccc atcctggaga gccccaccaa cctgtgaaac 4380

ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 4440 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 4440

aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 4500 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 4500

catgtctgg 4509 catgtctgg 4509

<210> 142 <210> 142 <211> 3155 <211> 3155 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="FLC‐2A7‐1" <223> /note="FLC-2A7-1"

<400> 142 <400> 142 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60

gctaaatcca aggtaaggtc agaagagcta gcgccaccat gctgctgctg gtgaccagcc 300 gctaaatcca aggtaaggtc agaagagcta gcgccaccat gctgctgctg gtgaccagcc 300

tgctgctgtg tgagctgccc caccccgcct ttctgctgat ccccgacatc cagatgaccc 360 tgctgctgtg tgagctgccc caccccgcct ttctgctgat ccccgacato cagatgacco 360

agaccacctc cagcctgagc gccagcctgg gcgaccgggt gaccatcagc tgccgggcca 420 agaccacctc cagcctgagc gccagcctgg gcgaccgggt gaccatcagc tgccgggcca 420

gccaggacat cagcaagtac ctgaactggt atcagcagaa gcccgacggc accgtcaagc 480 gccaggacat cagcaagtac ctgaactggt atcagcagaa gcccgacggc accgtcaagc 480

tgctgatcta ccacaccagc cggctgcaca gcggcgtgcc cagccggttt agcggcagcg 540 tgctgatcta ccacaccago cggctgcaca gcggcgtgcc cagccggttt agcggcagcg 540

gctccggcac cgactacagc ctgaccatct ccaacctgga gcaggaggac atcgccacct 600 gctccggcac cgactacagc ctgaccatct ccaacctgga gcaggaggad atcgccacct 600

Page 123 Page 123

50471‐706_601_SL.TXT acttttgcca gcagggcaac acactgccct acacctttgg cggcggaaca aagctggaga 660 099

tcaccggcag cacctccggc agcggcaagc ctggcagcgg cgagggcagc accaagggcg 720 OZL

aggtgaagct gcaggagagc ggccctggcc tggtggcccc cagccagagc ctgagcgtga 780 08L

cctgtaccgt gtccggcgtg tccctgcccg actacggcgt gtcctggatc cggcagcccc 840

ctaggaaggg cctggagtgg ctgggcgtga tctggggcag cgagaccacc tactacaaca 900 006

gcgccctgaa gagccggctg accatcatca aggacaacag caagagccag gtgttcctga 960 096

agatgaacag cctgcagacc gacgacaccg ccatctacta ctgtgccaag cactactact 1020

acggcggcag ctacgccatg gactactggg gccagggcac cagcgtgacc gtgtccagca 1080 080T

agcccaccac cacccctgcc cctagacctc caaccccagc ccctacaatc gccagccagc 1140

ccctgagcct gaggcccgaa gcctgtagac ctgccgctgg cggagccgtg cacaccagag 1200

gcctggattt cgcctgcgac atctacatct gggcccctct ggccggcacc tgtggcgtgc 1260 09 tgctgctgag cctggtcatc accctgtact gcaaccaccg gaataggagc aagcggagca 1320 OZET

gaggcggcca cagcgactac atgaacatga ccccccggag gcctggcccc acccggaagc 1380 08ET

actaccagcc ctacgcccct cccagggact tcgccgccta ccggagccgg gtgaagttca 1440

gccggagcgc cgacgcccct gcctaccagc agggccagaa ccagctgtac aacgagctga 1500 00ST

acctgggccg gagggaggag tacgacgtgc tggacaagcg gagaggccgg gaccctgaga 1560 09ST

tgggcggcaa gccccggaga aagaaccctc aggagggcct gtataacgaa ctgcagaaag 1620 029T the checked acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcggcgg aggggcaagg 1680 089T

gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggatacctac gacgccctgc 1740 STATE

acatgcaggc cctgcccccc agaagagcta agaggggaag cggagagggc agaggaagtc 1800 008T

tgctaacatg cggtgacgtc gaggagaatc ctggacctga ttggacctgg attctgtttc 1860 098T cheese tggtggccgc tgccacaaga gtgcacagca actgggtgaa tgtgatcagc gacctgaaga 1920 026T

agatcgagga tctgatccag agcatgcaca ttgatgccac cctgtacaca gaatctgatg 1980 086T

e e tgcaccctag ctgtaaagtg accgccatga agtgttttct gctggagctg caggtgattt 2040 9702

ctctggaaag cggagatgcc tctatccacg acacagtgga gaatctgatc atcctggcca 2100 00I2

acaatagcct gagcagcaat ggcaatgtga cagagtctgg ctgtaaggag tgtgaggagc 2160

Page 124

50471‐706_601_SL.TXT 50471-706_601_SL.TXT tggaggagaa gaacatcaag gagtttctgc agagctttgt gcacatcgtg cagatgttca 2220 tggaggagaa gaacatcaag gagtttctgc agagctttgt gcacatcgtg cagatgttca 2220

tcaatacaag ctctggcgga ggatctggag gaggcggatc tggaggagga ggcagtggag 2280 tcaatacaag ctctggcgga ggatctggag gaggcggatc tggaggagga ggcagtggag 2280

gcggaggatc tggcggagga tctctgcaga ttacatgccc tcctccaatg tctgtggagc 2340 gcggaggato tggcggagga tctctgcaga ttacatgccc tcctccaatg tctgtggagg 2340

acgccgatat ttgggtgaag tcctacagcc tgtacagcag agagagatac atctgcaaca 2400 acgccgatat ttgggtgaag tcctacagcc tgtacagcag agagagatad atctgcaaca 2400

gcggctttaa gagaaaggcc ggcacctctt ctctgacaga gtgcgtgctg aataaggcca 2460 gcggctttaa gagaaaggcc ggcacctctt ctctgacaga gtgcgtgctg aataaggcca 2460

caaatgtggc ccactggaca acacctagcc tgaagtgcat tagagatcct gccctggtcc 2520 caaatgtggc ccactggaca acacctagcc tgaagtgcat tagagatcct gccctggtcc 2520

accagaggcc tgcccctcca tctacagtga caacagccgg agtgacacct cagcctgaat 2580 accagaggcc tgcccctcca tctacagtga caacagccgg agtgacacct cagcctgaat 2580

ctctgagccc ttctggaaaa gaacctgccg ccagctctcc tagctctaat aataccgccg 2640 ctctgagccc ttctggaaaa gaacctgccg ccagctctcc tagctctaat aataccgccg 2640

ccacaacagc cgccattgtg cctggatctc agctgatgcc tagcaagtct cctagcacag 2700 ccacaacage cgccattgtg cctggatctc agctgatgcc tagcaagtct cctagcacag 2700

gcacaacaga gatcagcagc cacgaatctt ctcacggaac accttctcag accaccgcca 2760 gcacaacaga gatcagcage cacgaatctt ctcacggaac accttctcag accaccgcca 2760

agaattggga gctgacagcc tctgcctctc accagcctcc aggagtgtat cctcagggcc 2820 agaattggga gctgacagcc tctgcctctc accagcctcc aggagtgtat cctcagggcc 2820

actctgatac aacagtggcc atcagcacat ctacagtgct gctgtgtgga ctgtctgccg 2880 actctgatac aacagtggcc atcagcacat ctacagtgct gctgtgtgga ctgtctgccg 2880

tgtctctgct ggcctgttac ctgaagtcta gacagacacc tcctctggcc tctgtggaga 2940 tgtctctgct ggcctgttac ctgaagtcta gacagacacc tcctctggcc tctgtggaga 2940

tggaggccat ggaagccctg cctgtgacat ggggaacaag cagcagagat gaggacctgg 3000 tggaggccat ggaagccctg cctgtgacat ggggaacaag cagcagagat gaggacctgg 3000

agaattgttc tcaccacctg tgaaacttgt ttattgcagc ttataatggt tacaaataaa 3060 agaattgttc tcaccacctg tgaaacttgt ttattgcagc ttataatggt tacaaataaa 3060

gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 3120 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 3120

tgtccaaact catcaatgta tcttatcatg tctgg 3155 tgtccaaact catcaatgta tcttatcatg tctgg 3155

<210> 143 <210> 143 <211> 3476 <211> 3476 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="FLC‐2A5‐2" <223> /note="FLC-2A5-2"

<400> 143 <400> 143 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60 attgttcgga gcagtgcggc gcgtttagcg gagtactgtc ctccgatatt aatcggggca 60

Page 125 Page 125

50471‐706_601_SL.TXT 1x1'7S gactattccg gggtttaccg gcgcactctc gcccgaactt caccggcggt ctttcgtccg 120

tgctttatcg gggcggatca ctccgaaccc cgggaggtct atataagcag agctcgttta 180 08T

gtgaaccctc attctggaga cggatcccga gccgagtgtt ttgacctcca tagaacagcc 240

gctaaatcca aggtaaggtc agaagagcta gcgccaccat ggattggacc tggattctgt 300 00E

ttctggtggc cgctgccaca agagtgcaca gcaactgggt gaatgtgatc agcgacctga 360 09E

e agaagatcga ggatctgatc cagagcatgc acattgatgc caccctgtac acagaatctg 420

7 atgtgcaccc tagctgtaaa gtgaccgcca tgaagtgttt tctgctggag ctgcaggtga 480 08/

e tttctctgga aagcggagat gcctctatcc acgacacagt ggagaatctg atcatcctgg 540

ccaacaatag cctgagcagc aatggcaatg tgacagagtc tggctgtaag gagtgtgagg 600 009

agctggagga gaagaacatc aaggagtttc tgcagagctt tgtgcacatc gtgcagatgt 660 099

tcatcaatac aagctctggc ggaggatctg gaggaggcgg atctggagga ggaggcagtg 720 OZL

e gaggcggagg atctggcgga ggatctctgc agattacatg ccctcctcca atgtctgtgg 780 08L

agcacgccga tatttgggtg aagtcctaca gcctgtacag cagagagaga tacatctgca 840

acagcggctt taagagaaag gccggcacct cttctctgac agagtgcgtg ctgaataagg 900 006

e ccacaaatgt ggcccactgg acaacaccta gcctgaagtg cattagagat cctgccctgg 960 096

tccaccagag gcctgcccct ccatctacag tgacaacagc cggagtgaca cctcagcctg 1020

aatctctgag cccttctgga aaagaacctg ccgccagctc tcctagctct aataataccg 1080 080T

ccgccacaac agccgccatt gtgcctggat ctcagctgat gcctagcaag tctcctagca 1140

caggcacaac agagatcagc agccacgaat cttctcacgg aacaccttct cagaccaccg 1200

ccaagaattg ggagctgaca gcctctgcct ctcaccagcc tccaggagtg tatcctcagg 1260 092I

gccactctga tacaacagtg gccatcagca catctacagt gctgctgtgt ggactgtctg 1320 OZET

ccgtgtctct gctggcctgt tacctgaagt ctagacagac acctcctctg gcctctgtgg 1380 08ET

agatggaggc catggaagcc ctgcctgtga catggggaac aagcagcaga gatgaggacc 1440

tggagaattg ttctcaccac ctgtgaatcg attaatctag cggccctaga cgagcagaca 1500 00ST

the tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 1560 09ST

ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 1620 029T

Page 126 9T aged

50471‐706_601_SL.TXT 50471-706_601_SL.TXT aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg 1680 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg 1680

ttttttaaag caagtaaaac ctctacaaat gtggtaaaat ccgataagcg tacctagagg 1740 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat ccgataagcg tacctagagg 1740

ccgcctagag attcgtgggg gactcgagga taggcggcgg ttgggtgtgc gacatgtccg 1800 ccgcctagag attcgtgggg gactcgagga taggcggcgg ttgggtgtgc gacatgtccg 1800

ccacatccca gatctcctcg aggaaaggcg gcagctttct gttcttgagc ttgagggaga 1860 ccacatccca gatctcctcg aggaaaggcg gcagctttct gttcttgagc ttgagggaga 1860

tgcacatgtt ggagttttgc atgccgagcg tgcgtagctc agagaggatt gagaggatct 1920 tgcacatgtt ggagttttgc atgccgagcg tgcgtagctc agagaggatt gagaggatct 1920

tgccgtatat gacggacgaa cgcgccgacc cgctcagctg gttcaggata tagatgcgga 1980 tgccgtatat gacggacgaa cgcgccgacc cgctcagctg gttcaggata tagatgcgga 1980

gcgtattcag gtagtaccgc tggatctctt ccaccagttg cggctgctcc aaccctggcc 2040 gcgtattcag gtagtaccgc tggatctctt ccaccagttg cggctgctcc aaccctggcc 2040

ggtcagaaaa gatgacgaca gccgtgagca gcgcgtaatg gatgttgtcc aacgccatag 2100 ggtcagaaaa gatgacgaca gccgtgagca gcgcgtaatg gatgttgtcc aacgccatag 2100

agtacatgca ccggcagaag tgcagtagat cctcgatgac ctcggccatg ccagccttgc 2160 agtacatgca ccggcagaag tgcagtagat cctcgatgac ctcggccatg ccagccttgc 2160

ggtagttgtc gcgagtgtac gcttggttgt tcgcgaacag aatactgtct gaggccgcat 2220 ggtagttgtc gcgagtgtac gcttggttgt tcgcgaacag aatactgtct gaggccgcat 2220

cgtatcgtcg cgcgactcgg agcatcatta cctcacttga gcaagcctta agcagcgtaa 2280 cgtatcgtcg cgcgactcgg agcatcatta cctcacttga gcaagcctta agcagcgtaa 2280

tttgatcagg ctgcgagatc ttggcgaacc ctggcaatcc cttcgcgaac tccacgataa 2340 tttgatcagg ctgcgagatc ttggcgaacc ctggcaatcc cttcgcgaac tccacgataa 2340

gttggaccgt gaggatagtc atctctgtga tctggcggaa gggagtgtcc gactcttcgt 2400 gttggaccgt gaggatagtc atctctgtga tctggcggaa gggagtgtcc gactcttcgt 2400

tttcatcgtc cgcttgctgc cacgtctgcg taatcctctt caaatcttca tcagaaggct 2460 tttcatcgtc cgcttgctgc cacgtctgcg taatcctctt caaatcttca tcagaaggct 2460

gctcgtaccc gtcctggtac cagatgagcc tggcgataag gaactgctgg ttggctgtca 2520 gctcgtaccc gtcctggtac cagatgagcc tggcgataag gaactgctgg ttggctgtca 2520

actgggggat gtttttctgc cggtttgtca ccaacagctt gtcggagaga aaccttggga 2580 actgggggat gtttttctgc cggtttgtca ccaacagctt gtcggagaga aaccttggga 2580

ccacttcgtg aatccttgct gcttcaggag gtggaggttc acactgcata atgggcggca 2640 ccacttcgtg aatccttgct gcttcaggag gtggaggttc acactgcata atgggcggca 2640

tgtggtcgtc caccgtcgtc gtgctgacag gcagtttgtc cttctccttc tgtgctttct 2700 tgtggtcgtc caccgtcgtc gtgctgacag gcagtttgtc cttctccttc tgtgctttct 2700

tctctttccg cttcatggcg cactgagtct cgggtactac gcactcaggc cgcccgggaa 2760 tctctttccg cttcatggcg cactgagtct cgggtactac gcactcaggc cgcccgggaa 2760

actcggggct cacggtcagc tgcctctggc ccttgttgct gctctcctcg ctgctgctgg 2820 actcggggct cacggtcagc tgcctctggc ccttgttgct gctctcctcg ctgctgctgg 2820

tggcgctgat cctgtgctgc ctcagggtca ggggcatgtc ggtctccacg ctggccagcc 2880 tggcgctgat cctgtgctgc ctcagggtca ggggcatgtc ggtctccacg ctggccagcc 2880

tgtcggtcac ggcgtccttg ttcacgttgt cctgcacgaa caggccggtc agcagggcct 2940 tgtcggtcac ggcgtccttg ttcacgttgt cctgcacgaa caggccggtc agcagggcct 2940

tgatgtcttg caggctgtcc atcttcagga tcatgtccag gtcctccctg gggaagatca 3000 tgatgtcttg caggctgtcc atcttcagga tcatgtccag gtcctccctg gggaagatca 3000

gcaggaacag ctgctccagc ctctccagcc tgctctccac ctcggtcagg tgggccctgg 3060 gcaggaacag ctgctccagc ctctccagcc tgctctccac ctcggtcagg tgggccctgg 3060

tcagggggct cctcttggtc ttggggctgt atctgcactc ccagttgttc ttcaggcact 3120 tcagggggct cctcttggtc ttggggctgt atctgcactc ccagttgttc ttcaggcact 3120

tggcgcactt gggcttctcc ttgctgcact tcagcttctt cagcctgcag atgtcgcaag 3180 tggcgcactt gggcttctcc ttgctgcact tcagcttctt cagcctgcag atgtcgcaag 3180

Page 127 Page 127

50471‐706_601_SL.TXT 50471-706_601_SL.TXT cctgctcgat gctgctcagc agcttcatgg tggcccgaaa agcacacaat gcctgtgttc cctgctcgat gctgctcagc agcttcatgg tggcccgaaa agcacacaat gcctgtgttc 3240 3240

tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac tactgcactt atatacggtt tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac tactgcactt atatacggtt 3300 3300

ctcccccacc ctcgggaaaa aggcggagco agtacacgad atcactttcc cagtttacco ctcccccacc ctcgggaaaa aggcggagcc agtacacgac atcactttcc cagtttaccc 3360 3360

cgcgccacct tctctaggca ccggttcaat cgccgacccc ctccccccaa cttctcgggg cgcgccacct tctctaggca ccggttcaat cgccgacccc ctccccccaa cttctcgggg 3420 3420

actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agcctcacgo acgctc actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agcctcacgc acgctc 3476 3476

<210> 144 <210> 144 <211> 3503 <211> 3503 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="FLC‐2A6‐1" <223> /note="FLC-2A6-1"

<400> 144 <400> 144 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagago gcacatcgcc cacagtecco gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60 60

gagaagttgg ggggaggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt gagaagttgg ggggaggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt 120 120

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaaco aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 180

acagcagccg ctaaatccaa ggtaaggtca gaagagctag cgccaccatg ctgctgctgg acagcagccg ctaaatccaa ggtaaggtca gaagagctag cgccaccatg ctgctgctgg 300 300

tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatco tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatcc 360 360

agatgaccca gaccacctcc agcctgagcg ccagcctggg cgaccgggtg accatcagct agatgaccca gaccacctcc agcctgagcg ccagcctggg cgaccgggtg accatcagct 420 420 gccgggccag ccaggacato agcaagtacc tgaactggta tcagcagaag cccgacggca gccgggccag ccaggacatc agcaagtacc tgaactggta tcagcagaag cccgacggca 480 480 ccgtcaagct gctgatctac cacaccagco ggctgcacag cggcgtgccc agccggttta ccgtcaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc agccggttta 540 540

gcggcagcgg ctccggcacc gactacagcc tgaccatctc caacctggag caggaggaca gcggcagcgg ctccggcacc gactacagcc tgaccatctc caacctggag caggaggaca 600 600

tcgccaccta cttttgccag cagggcaaca cactgcccta cacctttggc ggcggaacaa tcgccaccta cttttgccag cagggcaaca cactgcccta cacctttggc ggcggaacaa 660 660

agctggagat caccggcago acctccggca gcggcaagcc tggcagcggc gagggcagca agctggagat caccggcagc acctccggca gcggcaagcc tggcagcggc gagggcagca 720 720

ccaagggcga ggtgaagctg caggagagcg gccctggcct ggtggccccc agccagagcc ccaagggcga ggtgaagctg caggagagcg gccctggcct ggtggccccc agccagagcc 780 780

Page 128 Page 128

50471‐706_601_SL.TXT 7S [09] 1X1 tgagcgtgac ctgtaccgtg tccggcgtgt ccctgcccga ctacggcgtg tcctggatcc 840

ggcagccccc taggaagggc ctggagtggc tgggcgtgat ctggggcagc gagaccacct 900 006

actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacagc aagagccagg 960 096

tgttcctgaa gatgaacagc ctgcagaccg acgacaccgc catctactac tgtgccaagc 1020 0201

actactacta cggcggcagc tacgccatgg actactgggg ccagggcacc agcgtgaccg 1080 080I

tgtccagcaa gcccaccacc acccctgccc ctagacctcc aaccccagcc cctacaatcg 1140

ccagccagcc cctgagcctg aggcccgaag cctgtagacc tgccgctggc ggagccgtgc 1200

acaccagagg cctggatttc gcctgcgaca tctacatctg ggcccctctg gccggcacct 1260 The gtggcgtgct gctgctgagc ctggtcatca ccctgtactg caaccaccgg aataggagca 1320 OZET

agcggagcag aggcggccac agcgactaca tgaacatgac cccccggagg cctggcccca 1380 08ET

cccggaagca ctaccagccc tacgcccctc ccagggactt cgccgcctac cggagccggg 1440

tgaagttcag ccggagcgcc gacgcccctg cctaccagca gggccagaac cagctgtaca 1500 00ST

acgagctgaa cctgggccgg agggaggagt acgacgtgct ggacaagcgg agaggccggg 1560 09ST

ggggcaaggg ccacgacggc ctgtaccagg gcctgagcac cgccaccaag gatacctacg 1740

acgccctgca catgcaggcc ctgcccccca gatgaatcga ttaatctagc ggccctagac 1800 008D

gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 1860 098T

aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 1920 026T

the gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga 1980 086T

tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc cgataagcgt 2040

acctagaggc tcacaggtgg tgagaacaat tctccaggtc ctcatctctg ctgcttgttc 2100 00I2

cccatgtcac aggcagggct tccatggcct ccatctccac agaggccaga ggaggtgtct 2160 0912

gtctagactt caggtaacag gccagcagag acacggcaga cagtccacac agcagcactg 2220 0222

tagatgtgct gatggccact gttgtatcag agtggccctg aggatacact cctggaggct 2280 0822

ggtgagaggc agaggctgtc agctcccaat tcttggcggt ggtctgagaa ggtgttccgt 2340 OTEL

Page 129

50471‐706_601_SL.TXT 50471-706_601_SL.TXT gagaagattc gtggctgctg atctctgttg tgcctgtgct aggagacttg ctaggcatca 2400 gagaagatto gtggctgctg atctctgttg tgcctgtgct aggagacttg ctaggcatca 2400

gctgagatcc aggcacaatg gcggctgttg tggcggcggt attattagag ctaggagagc 2460 gctgagatcc aggcacaatg gcggctgttg tggcggcggt attattagag ctaggagago 2460

tggcggcagg ttcttttcca gaagggctca gagattcagg ctgaggtgtc actccggctg 2520 tggcggcagg ttcttttcca gaagggctca gagattcagg ctgaggtgtc actccggctg 2520

ttgtcactgt agatggaggg gcaggcctct ggtggaccag ggcaggatct ctaatgcact 2580 ttgtcactgt agatggaggg gcaggcctct ggtggaccag ggcaggatct ctaatgcact 2580

tcaggctagg tgttgtccag tgggccacat ttgtggcctt attcagcacg cactctgtca 2640 tcaggctagg tgttgtccag tgggccacat ttgtggcctt attcagcacg cactctgtca 2640

gagaagaggt gccggccttt ctcttaaagc cgctgttgca gatgtatctc tctctgctgt 2700 gagaagaggt gccggccttt ctcttaaagc cgctgttgca gatgtatctc tctctgctgt 2700

acaggctgta ggacttcacc caaatatcgg cgtgctccac agacattgga ggagggcatg 2760 acaggctgta ggacttcacc caaatatcgg cgtgctccac agacattgga ggagggcatg 2760

taatctgcag agatcctccg ccagatcctc cgcctccact gcctcctcct ccagatccgc 2820 taatctgcag agatcctccg ccagatcctc cgcctccact gcctcctcct ccagatccgc 2820

ctcctccaga tcctccgcca gagcttgtat tgatgaacat ctgcacgatg tgcacaaagc 2880 ctcctccaga tcctccgcca gagcttgtat tgatgaacat ctgcacgatg tgcacaaagc 2880

tctgcagaaa ctccttgatg ttcttctcct ccagctcctc acactcctta cagccagact 2940 tctgcagaaa ctccttgatg ttcttctcct ccagctcctc acactcctta cagccagact 2940

ctgtcacatt gccattgctg ctcaggctat tgttggccag gatgatcaga ttctccactg 3000 ctgtcacatt gccattgctg ctcaggctat tgttggccag gatgatcaga ttctccactg 3000

tgtcgtggat agaggcatct ccgctttcca gagaaatcac ctgcagctcc agcagaaaac 3060 tgtcgtggat agaggcatct ccgctttcca gagaaatcac ctgcagctcc agcagaaaac 3060

acttcatggc ggtcacttta cagctagggt gcacatcaga ttctgtgtac agggtggcat 3120 acttcatggc ggtcacttta cagctagggt gcacatcaga ttctgtgtac agggtggcat 3120

caatgtgcat gctctggatc agatcctcga tcttcttcag gtcgctgatc acattcaccc 3180 caatgtgcat gctctggatc agatcctcga tcttcttcag gtcgctgatc acattcaccc 3180

agttgctgtg cactcttgtg gcagcggcca ccagaaacag aatccaggtc caatccatgg 3240 agttgctgtg cactcttgtg gcagcggcca ccagaaacag aatccaggtc caatccatgg 3240

tggcgctagc ccgaaaagca cacaatgctt ctatggaggt caaaacactc ggctcgggat 3300 tggcgctagc ccgaaaagca cacaatgctt ctatggaggt caaaacactc ggctcgggat 3300

ccgtctccag aatgagggtt cactaaacga gctctgctta tatagacctc ccggggttcg 3360 ccgtctccag aatgagggtt cactaaacga gctctgctta tatagacctc ccggggttcg 3360

gagtgatccg ccccgataaa gcacggacga aagaccgccg gtgaagttcg ggcgagagtg 3420 gagtgatccg ccccgataaa gcacggacga aagaccgccg gtgaagttcg ggcgagagtg 3420

cgccggtaaa ccccggaata gtctgccccg attaatatcg gaggacagta ctccgctaaa 3480 cgccggtaaa ccccggaata gtctgccccg attaatatcg gaggacagta ctccgctaaa 3480

cgcgccgcac tgctccgaac aat 3503 cgcgccgcac tgctccgaac aat 3503

<210> 145 <210> 145 <211> 4079 <211> 4079 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220>

Page 130 Page 130

50471‐706_601_SL.TXT <221> source ananas <IZZ> <223> /note="XON‐00(h)" (4) )00-NOX,==70u/ <EZZ>

<400> 145 STATE <00 gagcgtgcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 60 09

gagaagttgg ggggaggggg tcggcgattg aaccggtgcc tagagaaggt ggcgcggggt 120 ...........

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 08T

gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240 0807777727

acagcagccg ctaaatccaa ggtaaggtca gaagagctag cgccaccatg ctgctgctgg 300 00E

tgaccagcct gctgctgtgt gagctgcccc accccgcctt tctgctgatc cccgacatcc 360 09E

agatgaccca gaccacctcc agcctgagcg ccagcctggg cgaccgggtg accatcagct 420

7 gccgggccag ccaggacatc agcaagtacc tgaactggta tcagcagaag cccgacggca 480 08/

ccgtcaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc agccggttta 540

gcggcagcgg ctccggcacc gactacagcc tgaccatctc caacctggag caggaggaca 600 009

tcgccaccta cttttgccag cagggcaaca cactgcccta cacctttggc ggcggaacaa 660 099

e e agctggagat caccggcagc acctccggca gcggcaagcc tggcagcggc gagggcagca 720 02L

ccaagggcga ggtgaagctg caggagagcg gccctggcct ggtggccccc agccagagcc 780 08L

tgagcgtgac ctgtaccgtg tccggcgtgt ccctgcccga ctacggcgtg tcctggatcc 840

ggcagccccc taggaagggc ctggagtggc tgggcgtgat ctggggcagc gagaccacct 900 006

actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacagc aagagccagg 960 096

tgttcctgaa gatgaacagc ctgcagaccg acgacaccgc catctactac tgtgccaagc 1020 020T

actactacta cggcggcagc tacgccatgg actactgggg ccagggcacc agcgtgaccg 1080 080D

tgtccagcaa gcccaccacc acccctgccc ctagacctcc aaccccagcc cctacaatcg 1140

ccagccagcc cctgagcctg aggcccgaag cctgtagacc tgccgctggc ggagccgtgc 1200 0021

acaccagagg cctggatttc gcctgcgaca tctacatctg ggcccctctg gccggcacct 1260 097T

gtggcgtgct gctgctgagc ctggtcatca ccctgtactg caaccaccgg aataggagca 1320 OZET

agcggagcag aggcggccac agcgactaca tgaacatgac cccccggagg cctggcccca 1380 08ET

cccggaagca ctaccagccc tacgcccctc ccagggactt cgccgcctac cggagccggg 1440

Page 131 IET

50471‐706_601_SL.TXT tgaagttcag ccggagcgcc gacgcccctg cctaccagca gggccagaac cagctgtaca 1500 00ST

acgagctgaa cctgggccgg agggaggagt acgacgtgct ggacaagcgg agaggccggg 1560 09ST

ggggcaaggg ccacgacggc ctgtaccagg gcctgagcac cgccaccaag gatacctacg 1740

acgccctgca catgcaggcc ctgcccccca gaagagctaa gaggggaagc ggagagggca 1800 008T

e See gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggacctatg cttctcctgg 1860 098T

tgacaagcct tctgctctgt gagttaccac acccagcatt cctcctgatc ccacgcaaag 1920 026T

tgtgtaacgg aataggtatt ggtgaattta aagactcact ctccataaat gctacgaata 1980 086T

ttaaacactt caaaaactgc acctccatca gtggcgatct ccacatcctg ccggtggcat 2040 9702

ttaggggtga ctccttcaca catactcctc ctctggatcc acaggaactg gatattctga 2100 0012

aaaccgtaaa ggaaatcaca gggtttttgc tgattcaggc ttggcctgaa aacaggacgg 2160 0877111898 0912

acctccatgc ctttgagaac ctagaaatca tacgcggcag gaccaagcaa catggtcagt 2220 0222

tttctcttgc agtcgtcagc ctgaacataa catccttggg attacgctcc ctcaaggaga 2280 0822

the taagtgatgg agatgtgata atttcaggaa acaaaaattt gtgctatgca aatacaataa 2340 OTEL

actggaaaaa actgtttggg acctccggtc agaaaaccaa aattataagc aacagaggtg 2400

e aaaacagctg caaggccaca ggccaggtct gccatgcctt gtgctccccc gagggctgct 2460

ggggcccgga gcccagggac tgcgtctctt gccggaatgt cagccgaggc agggaatgcg 2520 0252

tggacaagtg caaccttctg gagggtgagc caagggagtt tgtggagaac tctgagtgca 2580 credit 0852

tacagtgcca cccagagtgc ctgcctcagg ccatgaacat cacctgcaca ggacggggac 2640

be cagacaactg tatccagtgt gcccactaca ttgacggccc ccactgcgtc aagacctgcc 2700 00L2

cggcaggagt catgggagaa aacaacaccc tggtctggaa gtacgcagac gccggccatg 2760 09/2

tgtgccacct gtgccatcca aactgcacct acggatgcac tgggccaggt cttgaaggct 2820 0282

gtccaacgaa tgggcctaag atcccgtcca tcgccactgg gatggtgggg gccctcctct 2880 0887

tgctgctggt ggtggccctg gggatcggcc tcttcatgga gggcagagga agtctgctaa 2940 797 catgcggtga cgtcgaggag aatcctggac ctatgggccc caagaagaaa aggaaggtgg 3000 000E

Page 132 ZET aged

e

50471‐706_601_SL.TXT 50471-706_601_SL.TXT ccccccccac cgacgtgagc ctgggcgacg agctgcacct ggacggcgag gacgtggcca 3060 ccccccccac cgacgtgagc ctgggcgacg agctgcacct ggacggcgag gacgtggcca 3060

tggcccacgc cgacgccctg gacgacttcg acctggacat gctgggcgac ggcgacagcc 3120 tggcccacgc cgacgccctg gacgacttcg acctggacat gctgggcgad ggcgacagco 3120

ccggccccgg cttcaccccc cacgacagcg ccccctacgg cgccctggac atggccgact 3180 ccggccccgg cttcaccccc cacgacagcg ccccctacgg cgccctggac atggccgact 3180

tcgagttcga gcagatgttc accgacgccc tgggcatcga cgagtacggc ggcgaattcg 3240 tcgagttcga gcagatgttc accgacgccc tgggcatcga cgagtacggc ggcgaattcg 3240

agatgcccgt ggacaggatt ctggaggccg aactcgccgt ggagcagaaa agcgaccagg 3300 agatgcccgt ggacaggatt ctggaggccg aactcgccgt ggagcagaaa agcgaccagg 3300

gcgtggaggg ccccggcgga accggcggca gcggcagcag ccccaacgac cccgtgacca 3360 gcgtggaggg ccccggcgga accggcggca gcggcagcag ccccaaccaa cccgtgacca 3360

acatctgcca ggccgccgac aagcagctgt tcaccctggt ggagtgggcc aagaggattc 3420 acatctgcca ggccgccgac aagcagctgt tcaccctggt ggagtgggcc aagaggatto 3420

cccacttcag cagcctgccc ctggacgacc aggtgatcct gctgagggcc ggatggaacg 3480 cccacttcag cagcctgccc ctggacgaco aggtgatcct gctgagggcc ggatggaacg 3480

agctgctgat cgccagcttc agccacagga gcatcgacgt gagggacggc atcctgctgg 3540 agctgctgat cgccagcttc agccacagga gcatcgacgt gagggacggo atcctgctgg 3540

ccaccggcct gcacgtccat aggaacagcg cccacagcgc cggagtgggc gccatcttcg 3600 ccaccggcct gcacgtccat aggaacagcg cccacagcgc cggagtgggc gccatcttcg 3600

acagggtgct gaccgagctg gtgagcaaga tgagggacat gaggatggac aagaccgagc 3660 acagggtgct gaccgagctg gtgagcaaga tgagggacat gaggatggad aagaccgago 3660

tgggctgcct gagggccatc atcctgttca accccgaggt gaggggcctg aaaagcgccc 3720 tgggctgcct gagggccatc atcctgttca accccgaggt gaggggcctg aaaagcgccc 3720

aggaggtgga gctgctgagg gagaaggtgt acgccgccct ggaggagtac accaggacca 3780 aggaggtgga gctgctgagg gagaaggtgt acgccgccct ggaggagtad accaggacca 3780

cccaccccga cgagcccggc agattcgcca agctgctgct gaggctgccc agcctgagga 3840 cccaccccga cgagcccggc agattcgcca agctgctgct gaggctgccc agcctgagga 3840

gcatcggcct gaagtgcctg gagcacctgt tcttcttcag gctgatcggc gacgtgccca 3900 gcatcggcct gaagtgcctg gagcacctgt tcttcttcag gctgatcggo gacgtgccca 3900

tcgacacctt cctgatggag atgctggaga gccccagcga cagctgaaac ttgtttattg 3960 tcgacacctt cctgatggag atgctggaga gccccagcga cagctgaaac ttgtttattg 3960

cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 4020 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 4020

tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgg 4079 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgg 4079

<210> 146 <210> 146 <211> 18 <211> 18 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic peptide" peptide"

<400> 146 <400> 146

Page 133 Page 133

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 1 5 10 15

Lys Gly Lys Gly

<210> 147 <210> 147 <211> 26 <211> 26 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Linker" <223> /note="Linker"

<400> 147 <400> 147 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln 20 25 20 25

<210> 148 <210> 148 <211> 3 <211> 3 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 148 <400> 148 Gly Ser Gly Gly Ser Gly 1 1

<210> 149 <210> 149 <211> 4 <211> 4

Page 134 Page 134

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 149 <400> 149 Ser Gly Ser Gly Ser Gly Ser Gly 1 1

<210> 150 <210> 150 <211> 15 <211> 15 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 150 <400> 150 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 1 5 10 15

<210> 151 <210> 151 <211> 4 <211> 4 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 151 <400> 151 Arg Ala Lys Arg Arg Ala Lys Arg Page 135 Page 135

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 1 1

<210> 152 <210> 152 <211> 28 <211> 28 <212> PRT <212> PRT <213> Foot‐and‐mouth disease virus <213> Foot-and-mouth disease virus

<220> <220> <221> source <221> source <223> /note="Fmdv" <223> /note="Fmdv"

<400> 152 <400> 152 Arg Ala Lys Arg Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Arg Ala Lys Arg Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu 1 5 10 15 1 5 10 15

Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 20 25 20 25

<210> 153 <210> 153 <211> 18 <211> 18 <212> PRT <212> PRT <213> Thosea asigna virus <213> Thosea asigna virus

<400> 153 <400> 153 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 1 5 10 15

Gly Pro Gly Pro

<210> 154 <210> 154 <211> 25 <211> 25 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Furin‐GSG‐T2A" <223> /note="Furin-GSG-T2A"

Page 136 Page 136

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<400> 154 <400> 154 Arg Ala Lys Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Arg Ala Lys Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys 1 5 10 15 1 5 10 15

Gly Asp Val Glu Glu Asn Pro Gly Pro Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 20 25

<210> 155 <210> 155 <211> 26 <211> 26 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Furin‐SGSG‐T2A" <223> /note="Furin-SGSG-T2A"

<400> 155 <400> 155 Arg Ala Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Arg Ala Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr 1 5 10 15 1 5 10 15

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 20 25

<210> 156 <210> 156 <211> 19 <211> 19 <212> PRT <212> PRT <213> Porcine teschovirus <213> Porcine teschovirus

<220> <220> <221> source <221> source <223> /note="Porcine teschovirus‐1 2A region (P2A)" <223> /note="Porcine teschovirus -1 2A region (P2A)"

<400> 156 <400> 156 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 1 5 10 15

Pro Gly Pro Pro Gly Pro

<210> 157 <210> 157 Page 137 Page 137

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <211> 22 <211> 22 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= 'Description of Artificial Sequence: Synthetic peptide" peptide"

<400> 157 <400> 157 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 1 5 10 15

Glu Glu Asn Pro Gly Pro Glu Glu Asn Pro Gly Pro 20 20

<210> 158 <210> 158 <211> 20 <211> 20 <212> PRT <212> PRT <213> Equine rhinitis A virus <213> Equine rhinitis A virus

<220> <220> <221> source <221> source <223> /note="Equine rhinitis A virus 2A region (E2A)" <223> /note="Equine rhinitis A virus 2A region (E2A)"

<400> 158 <400> 158 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 1 5 10 15

Asn Pro Gly Pro Asn Pro Gly Pro 20 20

<210> 159 <210> 159 <211> 22 <211> 22 <212> PRT <212> PRT <213> Foot‐and‐mouth disease virus <213> Foot-and-mouth disease virus

<220> <220> <221> source <221> source <223> /note="Foot‐and‐mouth disease virus 2A region (F2A)" <223> /note="Foot-and-mouth disease virus 2A region (F2A)"

<400> 159 <400> 159 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 1 5 10 15 Page 138 Page 138

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Glu Ser Asn Pro Gly Pro Glu Ser Asn Pro Gly Pro 20 20

<210> 160 <210> 160 <211> 31 <211> 31 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="FP2A" <223> /note="FP2A"

<400> 160 <400> 160 Arg Ala Lys Arg Ala Pro Val Lys Gln Gly Ser Gly Ala Thr Asn Phe Arg Ala Lys Arg Ala Pro Val Lys Gln Gly Ser Gly Ala Thr Asn Phe 1 5 10 15 1 5 10 15

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 30 20 25 30

<210> 161 <210> 161 <211> 8 <211> 8 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 161 <400> 161 Ala Pro Val Lys Gln Gly Ser Gly Ala Pro Val Lys Gln Gly Ser Gly 1 5 1 5

<210> 162 <210> 162 <211> 5 <211> 5 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence Page 139 Page 139

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<220> <220> <221> source <221> source <223> /note="Linker" <223> /note="Linker"

<400> 162 <400> 162 Ala Pro Val Lys Gln Ala Pro Val Lys Gln 1 5 1 5

<210> 163 <210> 163 <211> 22 <211> 22 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 163 <400> 163 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 1 5 10 15

Ala Phe Leu Leu Ile Pro Ala Phe Leu Leu Ile Pro 20 20

<210> 164 <210> 164 <211> 20 <211> 20 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

Page 140 Page 140

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <400> 164 <400> 164 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro 1 5 10 15 1 5 10 15

Gly Ser Ser Gly Gly Ser Ser Gly 20 20

<210> 165 <210> 165 <211> 18 <211> 18 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 165 <400> 165 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 1 5 10 15

His Ser His Ser

<210> 166 <210> 166 <211> 21 <211> 21 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 166 <400> 166 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 1 5 10 15

His Ala Ala Arg Pro His Ala Ala Arg Pro Page 141 Page 141

50471‐706_601_SL.TXT 50471-706 601_SL.TX 20 20

<210> 167 <210> 167 <211> 21 <211> 21 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic peptide" peptide""

<220> <220> <221> source <221> source <223> /note="TVB2(T21A)signal peptide" <223> /note="TVB2 (T21A)signal peptide"

<400> 167 <400> 167 Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 1 5 10 15

Asp His Ala Asp Ala Asp His Ala Asp Ala 20 20

<210> 168 <210> 168 <211> 24 <211> 24 <212> PRT <212> PRT <213> Homo sapiens <213> Homo sapiens

<400> 168 <400> 168 Met Lys Arg Phe Leu Phe Leu Leu Leu Thr Ile Ser Leu Leu Val Met Met Lys Arg Phe Leu Phe Leu Leu Leu Thr Ile Ser Leu Leu Val Met 1 5 10 15 1 5 10 15

Val Gln Ile Gln Thr Gly Leu Ser Val Gln Ile Gln Thr Gly Leu Ser 20 20

<210> 169 <210> 169 <211> 28 <211> 28 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic Page 142 Page 142

50471‐706_601_SL.TXT 50471-706_601_SL.TX peptide" peptide"

<400> 169 <400> 169 Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu 1 5 10 15 1 5 10 15

Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala 20 25 20 25

<210> 170 <210> 170 <211> 47 <211> 47 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note="Description of Artificial Sequence: Synthetic polypeptide" polypeptide"

<400> 170 <400> 170 Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 1 5 10 15 1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 20 25 30 20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 35 40 45

<210> 171 <210> 171 <211> 94 <211> 94 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

Page 143 Page 143

50471‐706_601_SL.TXT 50471-706_601_SL.TX

<400> 171 <400> 171 Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 1 5 10 15 1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala 20 25 30 20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys 35 40 45 35 40 45

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 50 55 60 50 55 60

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 65 70 75 80 70 75 80

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 85 90 85 90

<210> 172 <210> 172 <211> 141 <211> 141 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 172 <400> 172 Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 1 5 10 15 1 5 10 15

Page 144 Page 144

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala 65 70 75 80 70 75 80

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro 85 90 95 85 90 95

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 100 105 110 100 105 110

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 115 120 125 115 120 125

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 130 135 140 130 135 140

<210> 173 <210> 173 <211> 188 <211> 188 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 173 <400> 173 Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 1 5 10 15 1 5 10 15

Page 145 Page 145

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys 35 40 45 35 40 45

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly 115 120 125 115 120 125

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Thr Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Thr 130 135 140 130 135 140

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 145 150 155 160 145 150 155 160

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 165 170 175 165 170 175

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 180 185 180 185

<210> 174 <210> 174 <211> 28 <211> 28 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic peptide" peptide"

<220> <220> <221> source <221> source Page 146 Page 146

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <223> /note="CD8‐alpha TM domain" <223> /note="CD8-alpha TM domain"

<400> 174 <400> 174 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn 20 25 20 25

<210> 175 <210> 175 <211> 27 <211> 27 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 175 <400> 175 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 20 25

<210> 176 <210> 176 <211> 41 <211> 41 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 176 <400> 176 Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 1 5 10 15

Page 147 Page 147

50471‐706_601_SL.TXT 50471-706_601_SL.TX

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 35 40

<210> 177 <210> 177 <211> 112 <211> 112 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= Description of Artificial Sequence: Synthetic polypeptide" polypeptide"

<400> 177 <400> 177 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 100 105 110

Page 148 Page 148

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <210> 178 <210> 178 <211> 42 <211> 42 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 178 <400> 178 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 35 40

<210> 179 <210> 179 <211> 24 <211> 24 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note=" "Description of Artificial Sequence: Synthetic peptide" peptide"

<400> 179 <400> 179 Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val 1 5 10 15 1 5 10 15

Tyr Ile Asn Met Pro Gly Arg Gly Tyr Ile Asn Met Pro Gly Arg Gly 20 20

<210> 180 <210> 180 Page 149 Page 149

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <211> 52 <211> 52 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic polypeptide" polypeptide'

<400> 180 <400> 180 Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala 1 5 10 15 1 5 10 15

Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu 20 25 30 20 25 30

Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg 35 40 45 35 40 45

Pro Tyr Tyr Lys Pro Tyr Tyr Lys 50 50

<210> 181 <210> 181 <211> 86 <211> 86 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="VP16 activation domain" <223> /note="VP16 activation domain"

<400> 181 <400> 181 Gly Pro Lys Lys Lys Arg Lys Val Ala Pro Pro Thr Asp Val Ser Leu Gly Pro Lys Lys Lys Arg Lys Val Ala Pro Pro Thr Asp Val Ser Leu 1 5 10 15 1 5 10 15

Gly Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Gly Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala Met Ala His Ala 20 25 30 20 25 30 Page 150 Page 150

50471‐706_601_SL.TXT 50471-706_601_SL.TX

Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser 35 40 45 35 40 45

Pro Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Pro Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu 50 55 60 50 55 60

Asp Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Asp Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly 65 70 75 80 70 75 80

Ile Asp Glu Tyr Gly Gly Ile Asp Glu Tyr Gly Gly 85 85

<210> 182 <210> 182 <211> 235 <211> 235 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= 'Description of Artificial Sequence: Synthetic polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="Retinoid x receptor (RxR)" <223> /note="Retinoid X receptor (RxR)" "

<400> 182 <400> 182 Glu Met Pro Val Asp Arg Ile Leu Glu Ala Glu Leu Ala Val Glu Gln Glu Met Pro Val Asp Arg Ile Leu Glu Ala Glu Leu Ala Val Glu Gln 1 5 10 15 1 5 10 15

Lys Ser Asp Gln Gly Val Glu Gly Pro Gly Gly Thr Gly Gly Ser Gly Lys Ser Asp Gln Gly Val Glu Gly Pro Gly Gly Thr Gly Gly Ser Gly 20 25 30 20 25 30

Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp Lys 35 40 45 35 40 45

Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe Ser Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe Ser 50 55 60 50 55 60

Ser Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Ser Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp Asn 65 70 75 80 70 75 80

Page 151 Page 151

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Asp Val Arg Asp Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Asp Val Arg Asp 85 90 95 85 90 95

Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala His Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala His 100 105 110 100 105 110

Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val 115 120 125 115 120 125

Ser Lys Met Arg Asp Met Arg Met Asp Lys Thr Glu Leu Gly Cys Leu Ser Lys Met Arg Asp Met Arg Met Asp Lys Thr Glu Leu Gly Cys Leu 130 135 140 130 135 140

Arg Ala Ile Ile Leu Phe Asn Pro Glu Val Arg Gly Leu Lys Ser Ala Arg Ala Ile Ile Leu Phe Asn Pro Glu Val Arg Gly Leu Lys Ser Ala 145 150 155 160 145 150 155 160

Gln Glu Val Glu Leu Leu Arg Glu Lys Val Tyr Ala Ala Leu Glu Glu Gln Glu Val Glu Leu Leu Arg Glu Lys Val Tyr Ala Ala Leu Glu Glu 165 170 175 165 170 175

Tyr Thr Arg Thr Thr His Pro Asp Glu Pro Gly Arg Phe Ala Lys Leu Tyr Thr Arg Thr Thr His Pro Asp Glu Pro Gly Arg Phe Ala Lys Leu 180 185 190 180 185 190

Leu Leu Arg Leu Pro Ser Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu Leu Leu Arg Leu Pro Ser Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu 195 200 205 195 200 205

His Leu Phe Phe Phe Arg Leu Ile Gly Asp Val Pro Ile Asp Thr Phe His Leu Phe Phe Phe Arg Leu Ile Gly Asp Val Pro Ile Asp Thr Phe 210 215 220 210 215 220

Leu Met Glu Met Leu Glu Ser Pro Ser Asp Ser Leu Met Glu Met Leu Glu Ser Pro Ser Asp Ser 225 230 235 225 230 235

<210> 183 <210> 183 <211> 323 <211> 323 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> Page 152 Page 152

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <221> source <221> source <223> /note="VP16‐linker‐RxR" <223> /note="VP16-linker-RxR'

<400> 183 <400> 183 Gly Pro Lys Lys Lys Arg Lys Val Ala Pro Pro Thr Asp Val Ser Leu Gly Pro Lys Lys Lys Arg Lys Val Ala Pro Pro Thr Asp Val Ser Leu 1 5 10 15 1 5 10 15

Gly Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Gly Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala Met Ala His Ala 20 25 30 20 25 30

Ile Asp Glu Tyr Gly Gly Glu Phe Glu Met Pro Val Asp Arg Ile Leu Ile Asp Glu Tyr Gly Gly Glu Phe Glu Met Pro Val Asp Arg Ile Leu 85 90 95 85 90 95

Glu Ala Glu Leu Ala Val Glu Gln Lys Ser Asp Gln Gly Val Glu Gly Glu Ala Glu Leu Ala Val Glu Gln Lys Ser Asp Gln Gly Val Glu Gly 100 105 110 100 105 110

Pro Gly Gly Thr Gly Gly Ser Gly Ser Ser Pro Asn Asp Pro Val Thr Pro Gly Gly Thr Gly Gly Ser Gly Ser Ser Pro Asn Asp Pro Val Thr 115 120 125 115 120 125

Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val Glu Trp Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val Glu Trp 130 135 140 130 135 140

Ala Lys Arg Ile Pro His Phe Ser Ser Leu Pro Leu Asp Asp Gln Val Ala Lys Arg Ile Pro His Phe Ser Ser Leu Pro Leu Asp Asp Gln Val 145 150 155 160 145 150 155 160

Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser Phe Ser Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser Phe Ser 165 170 175 165 170 175

His Arg Ser Ile Asp Val Arg Asp Gly Ile Leu Leu Ala Thr Gly Leu His Arg Ser Ile Asp Val Arg Asp Gly Ile Leu Leu Ala Thr Gly Leu 180 185 190 180 185 190

Page 153 Page 153

50471‐706_601_SL.TXT 50471-706 601_SL.TXT His Val His Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala Ile Phe His Val His Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala Ile Phe 195 200 205 195 200 205

Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met Arg Met Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met Arg Met 210 215 220 210 215 220

Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Ile Leu Phe Asn Pro Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Ile Leu Phe Asn Pro 225 230 235 240 225 230 235 240

Glu Val Arg Gly Leu Lys Ser Ala Gln Glu Val Glu Leu Leu Arg Glu Glu Val Arg Gly Leu Lys Ser Ala Gln Glu Val Glu Leu Leu Arg Glu 245 250 255 245 250 255

Lys Val Tyr Ala Ala Leu Glu Glu Tyr Thr Arg Thr Thr His Pro Asp Lys Val Tyr Ala Ala Leu Glu Glu Tyr Thr Arg Thr Thr His Pro Asp 260 265 270 260 265 270

Glu Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ser Leu Arg Glu Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ser Leu Arg 275 280 285 275 280 285

Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe Phe Arg Leu Ile Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe Phe Arg Leu Ile 290 295 300 290 295 300

Gly Asp Val Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu Ser Pro Gly Asp Val Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu Ser Pro 305 310 315 320 305 310 315 320

Ser Asp Ser Ser Asp Ser

<210> 184 <210> 184 <211> 150 <211> 150 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= 'Description of Artificial Sequence: Synthetic polypeptide" polypeptide'

<220> <220> <221> source <221> source <223> /note="GAL4 DNA Binding Domain" <223> /note= "GAL4 DNA Binding Domain"

<400> 184 <400> 184 Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Page 154 Page 154

50471‐706_601_SL.TXT 50471-706_601_SL.TXT - 1 5 10 15 1 5 10 15

Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30 20 25 30

Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45 35 40 45

Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55 60 50 55 60

Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile 65 70 75 80 70 75 80

Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85 90 95 85 90 95

Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala 100 105 110 100 105 110

Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser 115 120 125 115 120 125

Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu 130 135 140 130 135 140

Thr Val Ser Pro Glu Phe Thr Val Ser Pro Glu Phe 145 150 145 150

<210> 185 <210> 185 <211> 336 <211> 336 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Ecdysone Receptor Ligand Binding Domain <223> /note="Ecdysone Receptor Ligand Binding Domain Page 155 Page 155

50471‐706_601_SL.TXT 50471-706_601_SL.TX ‐ VY variant (EcR)" - VY variant (EcR) "

<400> 185 <400> 185 Ile Arg Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Ile Arg Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg 1 5 10 15 1 5 10 15

Lys Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Lys Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr 20 25 30 20 25 30

Thr Thr Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Thr Thr Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro 35 40 45 35 40 45

Pro Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Pro Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser 50 55 60 50 55 60

Asp Lys Leu Leu Val Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Asp Lys Leu Leu Val Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr 65 70 75 80 70 75 80

Ala Asn Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Ala Asn Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly 85 90 95 85 90 95

Tyr Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Tyr Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp 100 105 110 100 105 110

Gln Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Gln Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln 115 120 125 115 120 125

Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala 130 135 140 130 135 140

Lys Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Lys Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr 145 150 155 160 145 150 155 160

Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg 165 170 175 165 170 175

Arg Tyr Asp Ala Ala Ser Asp Ser Ile Leu Phe Ala Asn Asn Gln Ala Arg Tyr Asp Ala Ala Ser Asp Ser Ile Leu Phe Ala Asn Asn Gln Ala 180 185 190 180 185 190

Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Glu Val Ile Glu Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Glu Val Ile Glu Page 156 Page 156

50471‐706_601_SL.TXT 50471-706_601_SL.TX) 195 200 205 195 200 205

Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn 210 215 220 210 215 220

Ile His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro Ile His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro 225 230 235 240 225 230 235 240

Gly Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Gly Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu 245 250 255 245 250 255

Asn Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Asn Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg 260 265 270 260 265 270

Ser Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Ser Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg 275 280 285 275 280 285

Thr Leu Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Thr Leu Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys 290 295 300 290 295 300

Asn Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Asn Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp 305 310 315 320 305 310 315 320

Met Ser His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu Met Ser His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 325 330 335 325 330 335

<210> 186 <210> 186 <211> 335 <211> 335 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Ecdysone Receptor Ligand Binding Domain <223> /note="Ecdysone Receptor Ligand Binding Domain ‐ VY variant (EcR)" - VY variant (ECR) "

<400> 186 <400> 186 Arg Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Arg Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Page 157 Page 157

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 1 5 10 15 1 5 10 15

Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr 20 25 30 20 25 30

Thr Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Thr Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro 35 40 45 35 40 45

Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp 50 55 60 50 55 60

Lys Leu Leu Val Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Lys Leu Leu Val Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala 65 70 75 80 70 75 80

Asn Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Asn Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr 85 90 95 85 90 95

Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln 100 105 110 100 105 110

Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile 115 120 125 115 120 125

Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys 130 135 140 130 135 140

Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu 145 150 155 160 145 150 155 160

Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Arg Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Arg 165 170 175 165 170 175

Tyr Asp Ala Ala Ser Asp Ser Ile Leu Phe Ala Asn Asn Gln Ala Tyr Tyr Asp Ala Ala Ser Asp Ser Ile Leu Phe Ala Asn Asn Gln Ala Tyr 180 185 190 180 185 190

Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Glu Val Ile Glu Asp Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Glu Val Ile Glu Asp 195 200 205 195 200 205

Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Ile Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Ile Page 158 Page 158

50471‐706_601_SL.TXT 50471-706_601 _SL.TXT 210 215 220 210 215 220

His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro Gly His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro Gly 225 230 235 240 225 230 235 240

Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn 245 250 255 245 250 255

Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser 260 265 270 260 265 270

Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr 275 280 285 275 280 285

Leu Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Leu Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn 290 295 300 290 295 300

Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met 305 310 315 320 305 310 315 320

Ser His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu Ser His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 325 330 335 325 330 335

<210> 187 <210> 187 <211> 488 <211> 488 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> / /note= Description of Artificial Sequence: Synthetic polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="GAL4‐Linker‐EcR" <223> /note="GAL4-Linker-EcR'

<400> 187 <400> 187 Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu 1 5 10 15 1 5 10 15

Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30 20 25 30 Page 159 Page 159

50471‐706_601_SL.TXT 50471-706_601_SL.TX

Thr Val Ser Pro Glu Phe Pro Gly Ile Arg Pro Glu Cys Val Val Pro Thr Val Ser Pro Glu Phe Pro Gly Ile Arg Pro Glu Cys Val Val Pro 145 150 155 160 145 150 155 160

Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys Ala Gln Lys Glu Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys Ala Gln Lys Glu 165 170 175 165 170 175

Lys Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp Asp His Met Pro Lys Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp Asp His Met Pro 180 185 190 180 185 190

Pro Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala Ala Arg Ile His Pro Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala Ala Arg Ile His 195 200 205 195 200 205

Glu Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu Val Thr Asn Arg Glu Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu Val Thr Asn Arg 210 215 220 210 215 220

Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln Phe Leu Ile Ala Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln Phe Leu Ile Ala 225 230 235 240 225 230 235 240 Page 160 Page 160

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp 245 250 255 245 250 255

Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn Glu Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn Glu 260 265 270 260 265 270

Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr 275 280 285 275 280 285

Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Gly Phe Ala Lys Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Gly Phe Ala Lys 290 295 300 290 295 300

Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu 305 310 315 320 305 310 315 320

Val Met Met Leu Arg Val Ala Arg Arg Tyr Asp Ala Ala Ser Asp Ser Val Met Met Leu Arg Val Ala Arg Arg Tyr Asp Ala Ala Ser Asp Ser 325 330 335 325 330 335

Ile Leu Phe Ala Asn Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ile Leu Phe Ala Asn Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys 340 345 350 340 345 350

Ala Gly Met Ala Glu Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys Ala Gly Met Ala Glu Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys 355 360 365 355 360 365

Met Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala Met Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala 370 375 380 370 375 380

Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val 385 390 395 400 385 390 395 400

Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu 405 410 415 405 410 415

Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile 420 425 430 420 425 430

Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn 435 440 445 435 440 445 Page 161 Page 161

50471‐706_601_SL.TXT 50471-706_601_SL.TX

Met Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu Met Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu 450 455 460 450 455 460

Glu Glu Ile Trp Asp Val Ala Asp Met Ser His Thr Gln Pro Pro Pro Glu Glu Ile Trp Asp Val Ala Asp Met Ser His Thr Gln Pro Pro Pro 465 470 475 480 465 470 475 480

Ile Leu Glu Ser Pro Thr Asn Leu Ile Leu Glu Ser Pro Thr Asn Leu 485 485

<210> 188 <210> 188 <211> 487 <211> 487 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 188 <400> 188 Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu 1 5 10 15 1 5 10 15

Page 162 Page 162

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Thr Val Ser Pro Glu Phe Pro Gly Arg Pro Glu Cys Val Val Pro Glu Thr Val Ser Pro Glu Phe Pro Gly Arg Pro Glu Cys Val Val Pro Glu 145 150 155 160 145 150 155 160

Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys Ala Gln Lys Glu Lys Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys Ala Gln Lys Glu Lys 165 170 175 165 170 175

Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp Asp His Met Pro Pro Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp Asp His Met Pro Pro 180 185 190 180 185 190

Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala Ala Arg Ile His Glu Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala Ala Arg Ile His Glu 195 200 205 195 200 205

Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu Val Thr Asn Arg Gln Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu Val Thr Asn Arg Gln 210 215 220 210 215 220

Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln Phe Leu Ile Ala Arg Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln Phe Leu Ile Ala Arg 225 230 235 240 225 230 235 240

Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp Leu Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp Leu 245 250 255 245 250 255

Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn Glu Glu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn Glu Glu 260 265 270 260 265 270

Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr Val Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr Val 275 280 285 275 280 285

Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Gly Phe Ala Lys Ile Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Gly Phe Ala Lys Ile 290 295 300 290 295 300

Page 163 Page 163

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Val 305 310 315 320 305 310 315 320

Met Met Leu Arg Val Ala Arg Arg Tyr Asp Ala Ala Ser Asp Ser Ile Met Met Leu Arg Val Ala Arg Arg Tyr Asp Ala Ala Ser Asp Ser Ile 325 330 335 325 330 335

Leu Phe Ala Asn Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Leu Phe Ala Asn Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ala 340 345 350 340 345 350

Gly Met Ala Glu Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys Met Gly Met Ala Glu Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys Met 355 360 365 355 360 365

Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala Val Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala Val 370 375 380 370 375 380

Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val Glu Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val Glu 385 390 395 400 385 390 395 400

Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu Asn Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu Asn 405 410 415 405 410 415

Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile Leu Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile Leu 420 425 430 420 425 430

Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn Met Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn Met 435 440 445 435 440 445

Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu Glu Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu Glu 450 455 460 450 455 460

Glu Ile Trp Asp Val Ala Asp Met Ser His Thr Gln Pro Pro Pro Ile Glu Ile Trp Asp Val Ala Asp Met Ser His Thr Gln Pro Pro Pro Ile 465 470 475 480 465 470 475 480

Leu Glu Ser Pro Thr Asn Leu Leu Glu Ser Pro Thr Asn Leu 485 485

<210> 189 <210> 189 <211> 335 <211> 335 <212> PRT <212> PRT Page 164 Page 164

50471‐706_601_SL.TXT 50471-706_601_SL.TX <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Truncated EGFR (huEGFRt) (Her1t)" <223> /note="Truncated EGFR (huEGFRt) (Her1t)' "

<400> 189 <400> 189 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile 20 25 30 20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 35 40 45 35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55 60 50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn 65 70 75 80 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg 85 90 95 85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile 100 105 110 100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val 115 120 125 115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp 130 135 140 130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn 145 150 155 160 145 150 155 160

Page 165 Page 165

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175 165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190 180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu 195 200 205 195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln 210 215 220 210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 225 230 235 240 225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro 245 250 255 245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr 260 265 270 260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His 275 280 285 275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro 290 295 300 290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala 305 310 315 320 305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 325 330 335 325 330 335

<210> 190 <210> 190 <211> 244 <211> 244 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic

Page 166 Page 166

50471‐706_601_SL.TXT 50471-706_601_SL.TX polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 1 (Her1 truncated design 1) <223> /note="EGFR truncated design 1 (Her1 truncated design 1) (HER1t1)" (HER1t1)

<400> 190 <400> 190 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175 165 170 175

Page 167 Page 167

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190 180 185 190

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 195 200 205 195 200 205

Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 210 215 220 210 215 220

Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 225 230 235 240 225 230 235 240

Ser Lys Arg Ser Ser Lys Arg Ser

<210> 191 <210> 191 <211> 257 <211> 257 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= 'Description of Artificial Sequence: Synthetic polypeptide" polypeptide

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 2 (Her1 truncated design 2) <223> /note="EGFR truncated design 2 (Her1 truncated design 2) (HER1t2)" (HER1t2)"

<400> 191 <400> 191 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 168 Page 168

50471‐706_601_SL.TXT 50471-706_601_SL.TXT - Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn 65 70 75 80 70 75 80

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Gly Gly Gly Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Gly Gly Gly 195 200 205 195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 210 215 220

Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 225 230 235 240 225 230 235 240

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 245 250 255 245 250 255

Ser Ser

Page 169 Page 169

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <210> 192 <210> 192 <211> 276 <211> 276 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 3 (Her1 truncated design 3) <223> /note="EGFR truncated design 3 (Her1 truncated design 3) (HER1t3)" (HER1t3)'

<400> 192 <400> 192 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 170 Page 170

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn 145 150 155 160 145 150 155 160

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 225 230 235 240

Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 245 250 255 245 250 255

Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 260 265 270 260 265 270

Ser Lys Arg Ser Ser Lys Arg Ser 275 275

<210> 193 <210> 193 <211> 295 <211> 295 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 4 (Her1 truncated design 4) <223> /note="EGFR truncated design 4 (Her1 truncated design 4) (HER1t4)" (HER1t4)" "

<400> 193 <400> 193 Page 171 Page 171

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 172 Page 172

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln 210 215 220 210 215 220

Arg Gly Pro Asp Asn Cys Ile Gln Gly Gly Gly Gly Ser Gly Gly Gly Arg Gly Pro Asp Asn Cys Ile Gln Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 245 250 255

Gly Ser Gly Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Gly Ser Gly Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly 260 265 270 260 265 270

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 275 280 285 275 280 285

Trp Val Arg Ser Lys Arg Ser Trp Val Arg Ser Lys Arg Ser 290 295 290 295

<210> 194 <210> 194 <211> 308 <211> 308 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 5 (Her1 truncated design 5) <223> /note="EGFR truncated design 5 (Her1 truncated design 5) (HER1t5)" (HER1t5)" "

<400> 194 <400> 194 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 173 Page 173

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55 60 50 55 60

Page 174 Page 174

50471‐706_601_SL.TXT 50471-706 601_SL.TXT His Cys Val Lys Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly His Cys Val Lys Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 260 265 270 260 265 270

Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 275 280 285 275 280 285

Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 290 295 300 290 295 300

Ser Lys Arg Ser Ser Lys Arg Ser 305 305

<210> 195 <210> 195 <211> 328 <211> 328 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 6 (Her1 truncated design 6) <223> /note="EGFR truncated design 6 (Her1 truncated design 6) (HER1t6)" (HER1t6)"

<400> 195 <400> 195 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 175 Page 175

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg 85 90 95 85 90 95

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Gly Gly Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Gly Gly 275 280 285 275 280 285

Page 176 Page 176

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Gly Ser Gly Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Gly Ser Gly Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly 290 295 300 290 295 300

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 305 310 315 320 305 310 315 320

Phe Trp Val Arg Ser Lys Arg Ser Phe Trp Val Arg Ser Lys Arg Ser 325 325

<210> 196 <210> 196 <211> 343 <211> 343 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 7 (Her1 truncated design 7) <223> /note="EGFR truncated design 7 (Her1 truncated design 7) (HER1t7)" (HER1t7)"

<400> 196 <400> 196 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 177 Page 177

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile 100 105 110 100 105 110

Page 178 Page 178

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Gly Gly Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Gly Gly Gly Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly 305 310 315 320 305 310 315 320

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 325 330 335 325 330 335

Trp Val Arg Ser Lys Arg Ser Trp Val Arg Ser Lys Arg Ser 340 340

<210> 197 <210> 197 <211> 242 <211> 242 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 8 (Her1 truncated design 8) <223> /note="EGFR truncated design 8 (Her1 truncated design 8) (HER1t8)" (HER1t8)" "

<400> 197 <400> 197 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 179 Page 179

Gly Gly Gly Ser Glu Ile Thr Leu Ile Ile Phe Gly Val Met Ala Gly Gly Gly Gly Ser Glu Ile Thr Leu Ile Ile Phe Gly Val Met Ala Gly 210 215 220 210 215 220

Val Ile Gly Thr Ile Leu Leu Ile Ser Tyr Gly Ile Arg Arg Gly Gly Val Ile Gly Thr Ile Leu Leu Ile Ser Tyr Gly Ile Arg Arg Gly Gly 225 230 235 240 225 230 235 240

Gly Ser Gly Ser

<210> 198 <210> 198 <211> 239 <211> 239 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source Page 180 Page 180

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <223> /note="EGFR truncated design 9 (Her1 truncated design 9) <223> /note="EGFR truncated design 9 (Her1 truncated design 9) (HER1t9)" (HER1t9)" "

<400> 198 <400> 198 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 181 Page 181

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 195 200 205 195 200 205

Gly Gly Gly Ser Ile Thr Leu Ile Ile Phe Gly Val Met Ala Gly Val Gly Gly Gly Ser Ile Thr Leu Ile Ile Phe Gly Val Met Ala Gly Val 210 215 220 210 215 220

Ile Gly Thr Ile Leu Leu Ile Ser Tyr Gly Ile Gly Gly Gly Ser Ile Gly Thr Ile Leu Leu Ile Ser Tyr Gly Ile Gly Gly Gly Ser 225 230 235 225 230 235

<210> 199 <210> 199 <211> 239 <211> 239 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 10 (Her1 truncated design 10) <223> /note="EGFR truncated design 10 (Her1 truncated design 10) (HER1t10)" (HER1t10)"

<400> 199 <400> 199 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 1 5 10 15

Page 182 Page 182

Ile Gly Thr Ile Leu Leu Ala Leu Leu Ile Trp Gly Gly Gly Ser Ile Gly Thr Ile Leu Leu Ala Leu Leu Ile Trp Gly Gly Gly Ser 225 230 235 225 230 235

<210> 200 <210> 200 <211> 237 <211> 237 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFR truncated design 11 (Her1 truncated design 11) <223> /note="EGFR truncated design 11 (Her1 truncated design 11) (HER1t11)" (HER1t11)" "

<400> 200 <400> 200 Page 183 Page 183

Page 184 Page 184

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Gly Gly Ser Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Gly Gly Ser Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr 210 215 220 210 215 220

Gly Val Ile Leu Thr Ala Leu Phe Leu Gly Gly Gly Ser Gly Val Ile Leu Thr Ala Leu Phe Leu Gly Gly Gly Ser 225 230 235 225 230 235

<210> 201 <210> 201 <211> 297 <211> 297 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 201 <400> 201 Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro 1 5 10 15 1 5 10 15

Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg 20 25 30 20 25 30

Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu 35 40 45 35 40 45

Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile 50 55 60 50 55 60

Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile 65 70 75 80 70 75 80

Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile 85 90 95 85 90 95

Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu 100 105 110 100 105 110

Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile Page 185 Page 185

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 115 120 125 115 120 125

Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser 130 135 140 130 135 140

His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro 145 150 155 160 145 150 155 160

Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175 165 170 175

Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly 180 185 190 180 185 190

Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile 195 200 205 195 200 205

Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys 210 215 220 210 215 220

Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile 225 230 235 240 225 230 235 240

Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro 245 250 255 245 250 255

Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu 260 265 270 260 265 270

Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser 275 280 285 275 280 285

Ser Pro Ile Glu Asn Asp Ser Ser Pro Ser Pro Ile Glu Asn Asp Ser Ser Pro 290 295 290 295

<210> 202 <210> 202 <211> 263 <211> 263 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

Page 186 Page 186

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic polypeptide" polypeptide'

<220> <220> <221> source <221> source <223> /note="Truncated CD20 design 1 (CD20t1) [CD20(M1‐E263]" <223> /note="Truncated CD20 design 1 (CD20t1) [CD20(M1-E263]" "

<400> 202 <400> 202 Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro 1 5 10 15 1 5 10 15

Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile 115 120 125 115 120 125

Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175 165 170 175 Page 187 Page 187

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Lys Asn Glu Glu Asp Ile Glu Lys Asn Glu Glu Asp Ile Glu 260 260

<210> 203 <210> 203 <211> 114 <211> 114 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 203 <400> 203 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 35 40 45

Page 188 Page 188

50471‐706_601_SL.TXT 50471-706_601_SL.TX

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 100 105 110

Thr Ser Thr Ser

<210> 204 <210> 204 <211> 237 <211> 237 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 204 <400> 204 Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val 1 5 10 15 1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30 20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45 35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60 50 55 60

Page 189 Page 189

50471‐706_601_SL.TXT 50471-706 601 SL.TXT - Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val 65 70 75 80 70 75 80

Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly 85 90 95 85 90 95

Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr 100 105 110 100 105 110

Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro 115 120 125 115 120 125

Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr 130 135 140 130 135 140

Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser 145 150 155 160 145 150 155 160

His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val 165 170 175 165 170 175

Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser 180 185 190 180 185 190

Leu Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Leu Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser 195 200 205 195 200 205

Val Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Val Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser 210 215 220 210 215 220

Ser Arg Asp Glu Asp Leu Glu Asn Cys Ser His His Leu Ser Arg Asp Glu Asp Leu Glu Asn Cys Ser His His Leu 225 230 235 225 230 235

<210> 205 <210> 205 <211> 395 <211> 395 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

Page 190 Page 190

50471‐706_601_SL.TXT 50471-706_601_SL.TXT polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="Membrane bound Interleukin‐15 with signal peptide" <223> /note="Membrane bound Interleukin- 15 with signal peptide"

<400> 205 <400> 205 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 1 5 10 15

His Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp His Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp 20 25 30 20 25 30

Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp 35 40 45 35 40 45

Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu 50 55 60 50 55 60

Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr 65 70 75 80 70 75 80

Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly 85 90 95 85 90 95

Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys 100 105 110 100 105 110

Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe 115 120 125 115 120 125

Ile Asn Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Asn Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln Ile Thr Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Gln Ile Thr 145 150 155 160 145 150 155 160

Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 165 170 175 165 170 175

Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Page 191 Page 191

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 180 185 190 180 185 190

Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 195 200 205 195 200 205

Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 210 215 220 210 215 220

Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr 225 230 235 240 225 230 235 240

Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu 245 250 255 245 250 255

Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala 260 265 270 260 265 270

Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr 275 280 285 275 280 285

Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser 290 295 300 290 295 300

Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln 305 310 315 320 305 310 315 320

Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile 325 330 335 325 330 335

Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu 340 345 350 340 345 350

Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu 355 360 365 355 360 365

Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg 370 375 380 370 375 380

Asp Glu Asp Leu Glu Asn Cys Ser His His Leu Asp Glu Asp Leu Glu Asn Cys Ser His His Leu Page 192 Page 192

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 385 390 395 385 390 395

<210> 206 <210> 206 <211> 335 <211> 335 <212> PRT <212> PRT <213> Mus sp. <213> Mus sp.

<220> <220> <221> source <221> source <223> /note="Murine Interleukin‐12 subunit beta (p40)" <223> /note="Murine Interleukin-12 subunit beta (p40) "

<400> 206 <400> 206 Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu 1 5 10 15 1 5 10 15

Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val 20 25 30 20 25 30

Val Glu Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Val Glu Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu 35 40 45 35 40 45

Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln 50 55 60 50 55 60

Arg His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys Arg His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys 65 70 75 80 70 75 80

Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr 85 90 95 85 90 95

Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp 100 105 110 100 105 110

Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys 115 120 125 115 120 125

Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln 130 135 140 130 135 140

Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro 145 150 155 160 145 150 155 160

Page 193 Page 193

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Asp Ser Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Asp Ser Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys 165 170 175 165 170 175

Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln 180 185 190 180 185 190

Glu Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Glu Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu 195 200 205 195 200 205

Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser 210 215 220 210 215 220

Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln 225 230 235 240 225 230 235 240

Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro 245 250 255 245 250 255

Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val 260 265 270 260 265 270

Arg Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Arg Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys 275 280 285 275 280 285

Asn Gln Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln Asn Gln Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln 290 295 300 290 295 300

Cys Lys Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn Cys Lys Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn 305 310 315 320 305 310 315 320

Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser 325 330 335 325 330 335

<210> 207 <210> 207 <211> 215 <211> 215 <212> PRT <212> PRT <213> Mus sp. <213> Mus sp.

<220> <220> <221> source <221> source Page 194 Page 194

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <223> /note="Murine Interleukin‐12 subunit alpha (p35)" <223> note="Murine Interleukin-1 subunit alpha (p35)" "

<400> 207 <400> 207 Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu Leu Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu Leu 1 5 10 15 1 5 10 15

Asn His Leu Ser Leu Ala Arg Val Ile Pro Val Ser Gly Pro Ala Arg Asn His Leu Ser Leu Ala Arg Val Ile Pro Val Ser Gly Pro Ala Arg 20 25 30 20 25 30

Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp Met Val Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp Met Val 35 40 45 35 40 45

Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala Glu Asp Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala Glu Asp 50 55 60 50 55 60

Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr 65 70 75 80 70 75 80

Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg 85 90 95 85 90 95

Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr 100 105 110 100 105 110

Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys 115 120 125 115 120 125

Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His 130 135 140 130 135 140

Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala Ile Asp Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala Ile Asp 145 150 155 160 145 150 155 160

Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg Gln Lys Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg Gln Lys 165 170 175 165 170 175

Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys Leu Cys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys Leu Cys 180 185 190 180 185 190

Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn Arg Val Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn Arg Val Page 195 Page 195

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 195 200 205 195 200 205

Met Gly Tyr Leu Ser Ser Ala Met Gly Tyr Leu Ser Ser Ala 210 215 210 215

<210> 208 <210> 208 <211> 535 <211> 535 <212> PRT <212> PRT <213> Mus sp. <213> Mus sp.

<400> 208 <400> 208 Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu 1 5 10 15 1 5 10 15

Page 196 Page 196

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser Gly Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser Gly 325 330 335 325 330 335

Gly Gly Gly Gly Gly Ser Arg Val Ile Pro Val Ser Gly Pro Ala Arg Gly Gly Gly Gly Gly Ser Arg Val Ile Pro Val Ser Gly Pro Ala Arg 340 345 350 340 345 350

Page 197 Page 197

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp Met Val Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp Met Val 355 360 365 355 360 365

Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala Glu Asp Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala Glu Asp 370 375 380 370 375 380

Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr 385 390 395 400 385 390 395 400

Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg 405 410 415 405 410 415

Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr 420 425 430 420 425 430

Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys 435 440 445 435 440 445

Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His 450 455 460 450 455 460

Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala Ile Asp Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala Ile Asp 465 470 475 480 465 470 475 480

Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg Gln Lys Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg Gln Lys 485 490 495 485 490 495

Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys Leu Cys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys Leu Cys 500 505 510 500 505 510

Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn Arg Val Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn Arg Val 515 520 525 515 520 525

Met Gly Tyr Leu Ser Ser Ala Met Gly Tyr Leu Ser Ser Ala 530 535 530 535

<210> 209 <210> 209 <211> 532 <211> 532 <212> PRT <212> PRT Page 198 Page 198

50471‐706_601_SL.TXT 50471-706_601_SL.TX <213> Homo sapiens <213> Homo sapiens

<220> <220> <221> source <221> source <223> /note="Human single chain IL‐12 (p40‐linker‐p35)" <223> /note="Human single chain IL -12 - (p40-linker-p35)" "

<400> 209 <400> 209 Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 1 5 10 15

Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 20 25 30

Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 35 40 45

Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60 50 55 60

Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 70 75 80

Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 85 90 95

Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110 100 105 110

Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 115 120 125

Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 130 135 140

Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 145 150 155 160

Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 165 170 175

Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Page 199 Page 199

50471‐706_601_SL.TXT 50471-706_601_SL.TX 180 185 190 180 185 190

Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205 195 200 205

Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220 210 215 220

Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235 240 225 230 235 240

Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 245 250 255

Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 260 265 270

Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 275 280 285

Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 290 295 300

Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310 315 320 305 310 315 320

Glu Trp Ala Ser Val Pro Cys Ser Gly Gly Gly Gly Gly Gly Ser Arg Glu Trp Ala Ser Val Pro Cys Ser Gly Gly Gly Gly Gly Gly Ser Arg 325 330 335 325 330 335

Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His 340 345 350 340 345 350

His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala 355 360 365 355 360 365

Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His 370 375 380 370 375 380

Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Page 200 Page 200

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 385 390 395 400 385 390 395 400

Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser 405 410 415 405 410 415

Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met 420 425 430 420 425 430

Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln 435 440 445 435 440 445

Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg 450 455 460 450 455 460

Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met 465 470 475 480 465 470 475 480

Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu 485 490 495 485 490 495

Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu 500 505 510 500 505 510

His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr 515 520 525 515 520 525

Leu Asn Ala Ser Leu Asn Ala Ser 530 530

<210> 210 <210> 210 <211> 473 <211> 473 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="CD19‐specific chimeric antigen receptor <223> /note="CD19-specific chimeric antigen receptor Page 201 Page 201

50471‐706_601_SL.TXT 50471-706_601_SL.TX (CD19‐CD8a‐CD28‐CD3z)" (CD19-CD8a-CD28-CD3z)" "

<400> 210 <400> 210 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110 100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125 115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 130 135 140 130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 145 150 155 160 145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175 165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190 180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Page 202 Page 202

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 195 200 205 195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220 210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 225 230 235 240 225 230 235 240

Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Val Thr Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 245 250 255

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 260 265 270

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 275 280 285

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 290 295 300 290 295 300

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn 305 310 315 320 305 310 315 320

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 325 330 335 325 330 335

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 340 345 350 340 345 350

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser 355 360 365 355 360 365

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 370 375 380 370 375 380

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 385 390 395 400 385 390 395 400

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Page 203 Page 203

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 405 410 415 405 410 415

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 420 425 430 420 425 430

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 435 440 445 435 440 445

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 450 455 460 450 455 460

Leu His Met Gln Ala Leu Pro Pro Arg Leu His Met Gln Ala Leu Pro Pro Arg 465 470 465 470

<210> 211 <210> 211 <211> 495 <211> 495 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 211 <400> 211 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Page 204 Page 204

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 65 70 75 80 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Lys Pro Thr Thr Thr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Lys Pro Thr Thr Thr 260 265 270 260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Page 205 Page 205

50471‐706_601_SL.TXT 50471-706_601_SL.TX 275 280 285 275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 305 310 315 320

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 325 330 335

Tyr Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Gly Gly His Ser Tyr Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Gly Gly His Ser 340 345 350 340 345 350

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 355 360 365 355 360 365

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg 370 375 380 370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 385 390 395 400 385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 405 410 415 405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 420 425 430 420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 435 440 445 435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 450 455 460 450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 465 470 475 480 465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Page 206 Page 206

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 485 490 495 485 490 495

<210> 212 <210> 212 <211> 120 <211> 120 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note= "Description of Artificial Sequence: Synthetic polypeptide" polypeptide

<220> <220> <221> source <221> source <223> /note="Anti‐CD19 monoclonal antibody clone FMC63 <223> /note="Anti-CD19 monoclonal antibody clone FMC63 variable heavy chain" variable heavy chain"

<400> 212 <400> 212 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40 45 35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 100 105 110

Gly Thr Ser Val Thr Val Ser Ser Gly Thr Ser Val Thr Val Ser Ser 115 120 115 120

<210> 213 <210> 213

Page 207 Page 207

50471‐706_601_SL.TXT 50471-706_601_SL.TX) <211> 245 <211> 245 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 213 <400> 213 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 1 5 10 15

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Page 208 Page 208

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 145 150 155 160 145 150 155 160

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205 195 200 205

Val Thr Val Ser Ser Val Thr Val Ser Ser 245 245

<210> 214 <210> 214 <211> 111 <211> 111 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="hM195 VL" <223> /note="hM195 VL"

<400> 214 <400> 214 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 20 25 30

Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 35 40 45 Page 209 Page 209

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 55 60 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 70 75 80

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 85 90 95

Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 100 105 110

<210> 215 <210> 215 <211> 116 <211> 116 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 215 <400> 215 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55 60 50 55 60

Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 70 75 80

Page 210 Page 210

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 85 90 95

Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 100 105 110

Thr Val Ser Ser Thr Val Ser Ser 115 115

<210> 216 <210> 216 <211> 242 <211> 242 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 216 <400> 216 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 35 40 45

Page 211 Page 211

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly 100 105 110 100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115 120 125 115 120 125

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val 130 135 140 130 135 140

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met 145 150 155 160 145 150 155 160

His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr 165 170 175 165 170 175

Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe Lys Ser Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe Lys Ser 180 185 190 180 185 190

Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr Met Glu Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr Met Glu 195 200 205 195 200 205

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 210 215 220

Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 225 230 235 240

Ser Ser Ser Ser

<210> 217 <210> 217 <211> 943 <211> 943 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source Page 212 Page 212

50471‐706_601_SL.TXT 50471-706_601_SL.T) <223> /note="EGFR variant III (EGFRvIII)" <223> /note="EGFR variant III (EGFRvIII)' "

<400> 217 <400> 217 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr 20 25 30 20 25 30

Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser 35 40 45 35 40 45

Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly 50 55 60 50 55 60

Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp 65 70 75 80 70 75 80

Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr 85 90 95 85 90 95

Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp 100 105 110 100 105 110

Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu 115 120 125 115 120 125

Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro 130 135 140 130 135 140

Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg 145 150 155 160 145 150 155 160

Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu 165 170 175 165 170 175

Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly 180 185 190 180 185 190

Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Page 213 Page 213

50471‐706_601_SL.TXT 50471-706 _601_SL. TXT 195 200 205 195 200 205

Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile 210 215 220 210 215 220

Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His 225 230 235 240 225 230 235 240

Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys 245 250 255 245 250 255

Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys 260 265 270 260 265 270

Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys 275 280 285 275 280 285

Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys 290 295 300 290 295 300

Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp 305 310 315 320 305 310 315 320

Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn 325 330 335 325 330 335

Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu 340 345 350 340 345 350

Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly 355 360 365 355 360 365

Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val 370 375 380 370 375 380

Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe 385 390 395 400 385 390 395 400

Met Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Met Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Page 214 Page 214

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 405 410 415 405 410 415

Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro 420 425 430 420 425 430

Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile 435 440 445 435 440 445

Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp 450 455 460 450 455 460

Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu 465 470 475 480 465 470 475 480

Arg Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Arg Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala 485 490 495 485 490 495

Tyr Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Tyr Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly 500 505 510 500 505 510

Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe 515 520 525 515 520 525

Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser 530 535 540 530 535 540

Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr 545 550 555 560 545 550 555 560

Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val 565 570 575 565 570 575

Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala 580 585 590 580 585 590

Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys 595 600 605 595 600 605

Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Page 215 Page 215

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 610 615 620 610 615 620

Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu 625 630 635 640 625 630 635 640

Met Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Met Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile 645 650 655 645 650 655

Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys 660 665 670 660 665 670

Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala 675 680 685 675 680 685

Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met 690 695 700 690 695 700

Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met 705 710 715 720 705 710 715 720

His Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp His Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp 725 730 735 725 730 735

Glu Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Glu Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro 740 745 750 740 745 750

Gln Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Gln Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu 755 760 765 755 760 765

Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp 770 775 780 770 775 780

Arg Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln 785 790 795 800 785 790 795 800

Arg Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Arg Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp 805 810 815 805 810 815

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Page 216 Page 216

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 820 825 830 820 825 830

Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu 835 840 845 835 840 845

Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr 850 855 860 850 855 860

Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val 865 870 875 880 865 870 875 880

Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His 885 890 895 885 890 895

Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys 900 905 910 900 905 910

Glu Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala 915 920 925 915 920 925

Glu Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala Glu Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala 930 935 940 930 935 940

<210> 218 <210> 218 <211> 116 <211> 116 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> /note=" Description of Artificial Sequence: Synthetic polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone 139 VH" <223> /note="Anti- EGFRVIII Clone 139 VH"

<400> 218 <400> 218 Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 20 25 30

Page 217 Page 217

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 85 90 95

Ala Gly Ser Ser Gly Trp Ser Glu Tyr Trp Gly Gln Gly Thr Leu Val Ala Gly Ser Ser Gly Trp Ser Glu Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 100 105 110

Thr Val Ser Ser Thr Val Ser Ser 115 115

<210> 219 <210> 219 <211> 107 <211> 107 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 219 <400> 219 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asn Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asn 20 25 30 20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 35 40 45

Page 218 Page 218

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Thr Gly Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Thr Gly 50 55 60 50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Ile Val Ser Ser Leu Gln Pro Ser Gly Ser Gly Thr Glu Phe Thr Leu Ile Val Ser Ser Leu Gln Pro 65 70 75 80 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His His Ser Tyr Pro Leu Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His His Ser Tyr Pro Leu 85 90 95 85 90 95

Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 100 105

<210> 220 <210> 220 <211> 120 <211> 120 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 220 <400> 220 Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala 1 5 10 15 1 5 10 15

Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg Lys Phe 20 25 30 20 25 30

Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val 35 40 45 35 40 45

Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val 50 55 60 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 70 75 80

Page 219 Page 219

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 85 90 95

Thr Arg Gly Tyr Ser Ser Thr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Thr Arg Gly Tyr Ser Ser Thr Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 100 105 110

Gly Thr Thr Val Thr Val Ser Ser Gly Thr Thr Val Thr Val Ser Ser 115 120 115 120

<210> 221 <210> 221 <211> 107 <211> 107 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone MR1 VL" <223> /note="Anti- EGFRVIII Clone MR1 VL "

<400> 221 <400> 221 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp Glu Lys Val Thr Ile Arg Cys Met Thr Ser Thr Asp Ile Asp Asp Asp 20 25 30 20 25 30

Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Phe Leu Ile 35 40 45 35 40 45

Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser 50 55 60 50 55 60

Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser Ser Gly Thr Gly Thr Asp Phe Val Phe Thr Ile Glu Asn Thr Leu Ser 65 70 75 80 70 75 80

Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Phe Asn Val Pro Leu Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Phe Asn Val Pro Leu 85 90 95 85 90 95

Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Page 220 Page 220

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 100 105 100 105

<210> 222 <210> 222 <211> 120 <211> 120 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 222 <400> 222 Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Ala 1 5 10 15 1 5 10 15

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 85 90 95

Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Thr Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 100 105 110

Gly Thr Thr Val Thr Val Ser Ser Gly Thr Thr Val Thr Val Ser Ser 115 120 115 120

<210> 223 <210> 223 <211> 107 <211> 107 Page 221 Page 221

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone MR1‐1 VL" <223> /note="Anti- - EGFRVIII Clone MR1-1 VL"

<400> 223 <400> 223 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu 85 90 95 85 90 95

Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys 100 105 100 105

<210> 224 <210> 224 <211> 120 <211> 120 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> Page 222 Page 222

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone humMR1‐1 VH" <223> /note="Anti- EGFRVIII Clone humMR1-1 VH"

<400> 224 <400> 224 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe 20 25 30 20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 35 40 45

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 85 90 95

Ala Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Ala Arg Gly Tyr Ser Pro Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 100 105 110

Gly Thr Met Val Thr Val Ser Ser Gly Thr Met Val Thr Val Ser Ser 115 120 115 120

<210> 225 <210> 225 <211> 107 <211> 107 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 225 <400> 225 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Page 223 Page 223

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 1 5 10 15 1 5 10 15

Asp Arg Val Thr Ile Thr Cys Met Thr Ser Thr Asp Ile Asp Asp Asp Asp Arg Val Thr Ile Thr Cys Met Thr Ser Thr Asp Ile Asp Asp Asp 20 25 30 20 25 30

Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile 35 40 45 35 40 45

Tyr Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly Tyr Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 50 55 60

Ser Gly Ser Gly Thr Asp Phe Ile Phe Thr Ile Ser Ser Leu Gln Pro Ser Gly Ser Gly Thr Asp Phe Ile Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu 85 90 95 85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 100 105

<210> 226 <210> 226 <211> 120 <211> 120 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 226 <400> 226 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 1 5 10 15

Page 224 Page 224

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Phe Tyr Ser Asp Asn Val Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Phe Tyr Ser Asp Asn Val 50 55 60 50 55 60

Ala Arg Gly Tyr Ser Pro Tyr Ser Phe Ala Met Asp Tyr Trp Gly Gln Ala Arg Gly Tyr Ser Pro Tyr Ser Phe Ala Met Asp Tyr Trp Gly Gln 100 105 110 100 105 110

Gly Thr Met Val Thr Val Ser Ser Gly Thr Met Val Thr Val Ser Ser 115 120 115 120

<210> 227 <210> 227 <211> 107 <211> 107 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti‐ EGFRvIII Clone humMR1‐2 VL" <223> /note="Anti- EGFRVIII Clone humMR1-2 VL" "

<400> 227 <400> 227 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 225 Page 225

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

<210> 228 <210> 228 <211> 241 <211> 241 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 228 <400> 228 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 226 Page 226

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly 100 105 110 100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln 115 120 125 115 120 125

Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 130 135 140 130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 145 150 155 160 145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile 165 170 175 165 170 175

Ser Gly Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Ser Gly Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 195 200 205 195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Ser Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Ser 210 215 220 210 215 220

Ser Gly Trp Ser Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Trp Ser Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 225 230 235 240 225 230 235 240

Ser Ser

<210> 229 <210> 229 <211> 242 <211> 242 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source Page 227 Page 227

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <223> /note="Anti‐EGFRvIII scFv clone MR1" <223> note="Anti-EGFRvIII scFv clone MR1"

<400> 229 <400> 229 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Lys Leu Gln Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Lys Leu Gln Gln 115 120 125 115 120 125

Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Ser Leu Lys Leu Ser Cys Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Ser Leu Lys Leu Ser Cys 130 135 140 130 135 140

Val Thr Ser Gly Phe Thr Phe Arg Lys Phe Gly Met Ser Trp Val Arg Val Thr Ser Gly Phe Thr Phe Arg Lys Phe Gly Met Ser Trp Val Arg 145 150 155 160 145 150 155 160

Gln Thr Ser Asp Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Thr Gly Gln Thr Ser Asp Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Thr Gly 165 170 175 165 170 175

Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr Ile Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr Ile 180 185 190 180 185 190

Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Page 228 Page 228

50471‐706_601_SL.TXT 50471-706_601_SL.TXT 195 200 205 195 200 205

Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Arg Gly Tyr Ser Ser Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Arg Gly Tyr Ser Ser 210 215 220 210 215 220

Thr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Thr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 225 230 235 240 225 230 235 240

Ser Ser Ser Ser

<210> 230 <210> 230 <211> 242 <211> 242 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Anti EGFRvIII scFv clone MR1‐1" <223> /note="Anti EGFRVIII scFv clone MR1-1"

<400> 230 <400> 230 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu Glu Asp Val Gly Asp Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu 85 90 95 85 90 95 Page 229 Page 229

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu 195 200 205 195 200 205

Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Arg Gly Tyr Ser Pro Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Arg Gly Tyr Ser Pro 210 215 220 210 215 220

Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 225 230 235 240 225 230 235 240

Ser Ser Ser Ser

<210> 231 <210> 231 <211> 242 <211> 242 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

Page 230 Page 230

50471‐706_601_SL.TXT 50471-706_601_SL.TXT <220> <220> <221> source <221> source <223> /note="Anti‐EGFRvIII scFv clone huMR1‐1" <223> /note="Anti-EGFRvIII - scFv clone huMR1-1"

<400> 231 <400> 231 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu 115 120 125 115 120 125

Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys 130 135 140 130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg 145 150 155 160 145 150 155 160

Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Thr Gly Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Thr Gly 165 170 175 165 170 175

Page 231 Page 231

50471‐706_601_SL.TXT 50471-706_601_SL.TXT

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu 195 200 205 195 200 205

Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Tyr Ser Pro Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Tyr Ser Pro 210 215 220 210 215 220

Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val 225 230 235 240 225 230 235 240

Ser Ser Ser Ser

<210> 232 <210> 232 <211> 242 <211> 242 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<400> 232 <400> 232 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 232 Page 232

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser Trp Asn Val Pro Leu 85 90 95 85 90 95

Gly Tyr Asn Thr Phe Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr Ile Gly Tyr Asn Thr Phe Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr Ile 180 185 190 180 185 190

Tyr Ser Phe Ala Met Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Tyr Ser Phe Ala Met Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val 225 230 235 240 225 230 235 240

Ser Ser Ser Ser

<210> 233 <210> 233 <211> 470 <211> 470 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

Page 233 Page 233

50471‐706_601_SL.TXT 50471-706_601_SL.TX polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (clone 139 scFv.CD8alpha hinge <223> /note=' 'EGFRVIII CAR (clone 139 scFv. CD8alpha hinge &TM.4‐1BB.CD3‐zeta)" &TM.4-1BB.CD3-zeta) "

<400> 233 <400> 233 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly 100 105 110 100 105 110

Page 234 Page 234

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Ser Gly Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Ser Gly Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 180 185 190

Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 245 250 255 245 250 255

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 260 265 270 260 265 270

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 275 280 285 275 280 285

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 290 295 300 290 295 300

Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg 305 310 315 320 305 310 315 320

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 325 330 335 325 330 335

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 340 345 350 340 345 350

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365 355 360 365

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380 370 375 380

Page 235 Page 235

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 385 390 395 400 385 390 395 400

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 405 410 415 405 410 415

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 420 425 430 420 425 430

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 435 440 445 435 440 445

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 450 455 460 450 455 460

Gln Ala Leu Pro Pro Arg Gln Ala Leu Pro Pro Arg 465 470 465 470

<210> 234 <210> 234 <211> 471 <211> 471 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (MR1 scFv.CD8alpha hinge <223> /note=' 'EGFRVIII CAR (MR1 scFv. CD8alpha hinge &TM.4‐1BB.CD3‐zeta)" &TM.4-1BB.CD3-zeta)" "

<400> 234 <400> 234 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Page 236 Page 236

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser 50 55 60 50 55 60

Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 245 250 255 245 250 255

Page 237 Page 237

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 260 265 270 260 265 270

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 275 280 285 275 280 285

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 290 295 300 290 295 300

Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly 305 310 315 320 305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 325 330 335 325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 340 345 350 340 345 350

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 355 360 365 355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 370 375 380 370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 385 390 395 400 385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 405 410 415 405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 420 425 430 420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 435 440 445 435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 450 455 460 450 455 460

Page 238 Page 238

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Met Gln Ala Leu Pro Pro Arg Met Gln Ala Leu Pro Pro Arg 465 470 465 470

<210> 235 <210> 235 <211> 471 <211> 471 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="Description of Artificial Sequence: Synthetic <223> (note= "Description of Artificial Sequence: Synthetic polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (MR1‐1 scFv.CD8alpha hinge <223> /note="EGFRVIII CAR (MR1-1 scFv.CD8alpha hinge &TM.4‐1BB.CD3‐zeta)" &TM.4-1BB.CD3-zeta) "

<400> 235 <400> 235 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Page 239 Page 239

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Ser Leu Lys Leu Ser Cys Ser Gly Gly Gly Leu Val Lys Pro Gly Ala Ser Leu Lys Leu Ser Cys 130 135 140 130 135 140

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 260 265 270 260 265 270

Page 240 Page 240

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 340 345 350 340 345 350

Met Gln Ala Leu Pro Pro Arg Met Gln Ala Leu Pro Pro Arg 465 470 465 470

<210> 236 <210> 236 <211> 471 <211> 471 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (humMR1‐1 scFv.CD8alpha hinge <223> /note= EGFRVIII CAR (humMR1-1 scFv. CD8alpha hinge &TM.4‐1BB.CD3‐zeta)" &TM. 4-1BB.CD3-zeta) "

<400> 236 <400> 236 Page 241 Page 241

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 242 Page 242

50471‐706_601_SL.TXT 50471-706_601_SL. TXT Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Tyr Ser Pro Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Tyr Ser Pro 210 215 220 210 215 220

Page 243 Page 243

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 420 425 430 420 425 430

Met Gln Ala Leu Pro Pro Arg Met Gln Ala Leu Pro Pro Arg 465 470 465 470

<210> 237 <210> 237 <211> 471 <211> 471 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (humMR1‐2 scFv.CD8alpha hinge <223> /note="EGFRvIII CAR (humMR1-2 scFv. CD8alpha hinge &TM.4‐1BB.CD3‐zeta)" &TM.4-1BB.CD3-zeta)"

<400> 237 <400> 237 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 244 Page 244

Page 245 Page 245

50471‐706_601_SL.TXT 50471-706 601SL.TXT Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 290 295 300 290 295 300

Met Gln Ala Leu Pro Pro Arg Met Gln Ala Leu Pro Pro Arg 465 470 465 470

<210> 238 <210> 238 <211> 518 <211> 518 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

Page 246 Page 246

50471‐706_601_SL.TXT 50471-706_601_SL.TX

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (MR1‐1 scFv.CD8alpha 2x hinge <223> /note= EGFRVIII CAR (MR1-1 scFv. CD8alpha 2x hinge &TM.4‐1BB.CD3‐zeta)" &TM. 4-1BB.CD3-zeta)" "

<400> 238 <400> 238 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Page 247 Page 247

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gln Thr Ser Asp Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Thr Gly Gln Thr Ser Asp Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Thr Gly 165 170 175 165 170 175

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg 260 265 270 260 265 270

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser 275 280 285 275 280 285

Asp Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Asp Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 290 295 300 290 295 300

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 305 310 315 320 305 310 315 320

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 325 330 335 325 330 335

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 340 345 350 340 345 350

Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg 355 360 365 355 360 365

Page 248 Page 248

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 370 375 380 370 375 380

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 385 390 395 400 385 390 395 400

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 405 410 415 405 410 415

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 420 425 430 420 425 430

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 435 440 445 435 440 445

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 450 455 460 450 455 460

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 465 470 475 480 465 470 475 480

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 485 490 495 485 490 495

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 500 505 510 500 505 510

Gln Ala Leu Pro Pro Arg Gln Ala Leu Pro Pro Arg 515 515

<210> 239 <210> 239 <211> 565 <211> 565 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source Page 249 Page 249

50471‐706_601_SL.TXT 50471-706_601_SL. TXT <223> /note="EGFRvIII CAR (MR1‐1 scFv.CD8alpha 3x hinge <223> /note= 'EGFRVIII CAR (MR1-1 scFv.CD8alpha 3x hinge &TM.4‐1BB.CD3‐zeta)" &TM. 4-1BB.CD3-zeta)'

<400> 239 <400> 239 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Page 250 Page 250

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu 195 200 205 195 200 205

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro 305 310 315 320 305 310 315 320

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp 325 330 335 325 330 335

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 340 345 350 340 345 350

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 355 360 365 355 360 365

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 370 375 380 370 375 380

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 385 390 395 400 385 390 395 400

Page 251 Page 251

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg Lys Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg Lys 405 410 415 405 410 415

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 420 425 430 420 425 430

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 435 440 445 435 440 445

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 450 455 460 450 455 460

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 465 470 475 480 465 470 475 480

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 485 490 495 485 490 495

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 500 505 510 500 505 510

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 515 520 525 515 520 525

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 530 535 540 530 535 540

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 545 550 555 560 545 550 555 560

Ala Leu Pro Pro Arg Ala Leu Pro Pro Arg 565 565

<210> 240 <210> 240 <211> 612 <211> 612 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

Page 252 Page 252

50471‐706_601_SL.TXT 50471-706_601_SL.TX) polypeptide" polypeptide"

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (MR1‐1 scFv.CD8alpha 4x hinge <223> /note=' 'EGFRVIII CAR (MR1-1 scFv. CD8alpha 4x hinge &TM.4‐1BB.CD3‐zeta)" &TM. .4-1BB.CD3-zeta) "

<400> 240 <400> 240 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ala Thr Gly 1 5 10 15 1 5 10 15

Page 253 Page 253

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr Ile Gly Tyr Asn Thr Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr Ile 180 185 190 180 185 190

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala 355 360 365 355 360 365

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys 370 375 380 370 375 380

Page 254 Page 254

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 385 390 395 400 385 390 395 400

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 405 410 415 405 410 415

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 420 425 430 420 425 430

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 435 440 445 435 440 445

Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg Lys Lys Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg Lys Lys 450 455 460 450 455 460

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 465 470 475 480 465 470 475 480

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 485 490 495 485 490 495

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 500 505 510 500 505 510

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 515 520 525 515 520 525

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 530 535 540 530 535 540

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 545 550 555 560 545 550 555 560

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 565 570 575 565 570 575

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 580 585 590 580 585 590

Page 255 Page 255

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 595 600 605 595 600 605

Leu Pro Pro Arg Leu Pro Pro Arg 610 610

<210> 241 <210> 241 <211> 565 <211> 565 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (huMR1‐1 scFv.CD8alpha 3x hinge <223> /note= 'EGFRVIII CAR (huMR1-1 scFv. CD8alpha 3x hinge & TM.4‐1BB.CD3‐zeta)" & TM. .4-1BB.CD3-zeta) "

<400> 241 <400> 241 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 256 Page 256

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu 115 120 125 115 120 125

Page 257 Page 257

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp 325 330 335 325 330 335

Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg Lys Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Lys Arg Gly Arg Lys 405 410 415 405 410 415

Page 258 Page 258

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 530 535 540 530 535 540

Ala Leu Pro Pro Arg Ala Leu Pro Pro Arg 565 565

<210> 242 <210> 242 <211> 612 <211> 612 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (huMR1‐1 scFv.CD8alpha 4x hinge <223> /note= "EGFRVIII CAR (huMR1-1 scFv. CD8alpha 4x hinge & TM.4‐1BB.CD3‐zeta)" & TM.4-1BB.CD3-zeta) "

<400> 242 <400> 242 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 259 Page 259

50471‐706_601_SL.TXT 50471-706_601_L.TXT Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 100 105 110

Page 260 Page 260

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro 305 310 315 320 305 310 315 320

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 385 390 395 400 385 390 395 400

Page 261 Page 261

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 515 520 525 515 520 525

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 595 600 605 595 600 605

Leu Pro Pro Arg Leu Pro Pro Arg 610 610

<210> 243 <210> 243 <211> 565 <211> 565 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (huMR1‐2 scFv.CD8alpha 3x hinge <223> /note= 'EGFRVIII CAR (huMR1-2 scFv. CD8alpha 3x hinge & TM.4‐1BB.CD3‐zeta)" & TM. 4-1BB.CD3-zeta) - "

<400> 243 <400> 243 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 262 Page 262

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile 35 40 45 35 40 45

Page 263 Page 263

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 245 250 255 245 250 255

Page 264 Page 264

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 450 455 460 450 455 460

Ala Leu Pro Pro Arg Ala Leu Pro Pro Arg 565 565

<210> 244 <210> 244 <211> 612 <211> 612 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <221> source <221> source <223> /note="EGFRvIII CAR (huMR1‐2 scFv.CD8alpha 4x hinge <223> /note= 'EGFRVIII CAR (huMR1-2 scFv. CD8alpha 4x hinge & TM.4‐1BB.CD3‐zeta" & TM. 4-1BB.CD3-zeta" -

<400> 244 <400> 244 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15

Page 265 Page 265

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Asp Arg Val Thr Ile Thr Cys Met Thr Ser Thr Asp Ile Asp Asp Asp Asp Arg Val Thr Ile Thr Cys Met Thr Ser Thr Asp Ile Asp Asp Asp 20 25 30 20 25 30

Page 266 Page 266

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Tyr Ser Phe Ala Met Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Tyr Ser Phe Ala Met Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val 225 230 235 240 225 230 235 240

Page 267 Page 267

50471‐706_601_SL.TXT 50471-706_601_SL.TXT Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 435 440 445 435 440 445

Leu Pro Pro Arg Leu Pro Pro Arg 610 610

Page 268 Page 268

Claims

CLAIMS WHAT IS CLAIMED IS: 1. A method for integrating a transgene into the genome of an isolated cell ex vivo, the method comprising delivering to the cell ex vivo: (a) a non-naturally occurring polynucleotide comprising (i) an AttP or AttB recombination sequence, and (ii) a transgene encoding a chimeric antigen receptor or a T-cell receptor; and (b) a polynucleotide encoding an SF370, SP c2, Bxbl, Al18, or <Rvl serine recombinase.
2. The method of claim 1, wherein the cell is an immune effector cell.
3. The method of claim 1, wherein the cell is a T cell or a NK cell.
4. The method of claim 1, wherein the cell is a peripheral blood mononuclear cell.
5. The method of claim 1, wherein the non-naturally occurring polynucleotide comprises an AttP recombination sequence.
6. The method of claim 1, wherein the non-naturally occurring polynucleotide comprises an AttB recombination sequence.
7. The method of claim 1, wherein the serine recombinase is an SF370 recombinase.
8. The method of claim 1, wherein the serine recombinase is an SP c2 recombinase.
9. The method of claim 1, wherein the polynucleotides are delivered by transfection, transformation, nucleofection, or transduction.
10. The method of claim 1, wherein the polynucleotides are delivered by calcium phosphate precipitation, lipofection, particle bombardment, or microinjection.
11. The method of claim 1, wherein the polynucleotides are delivered by electroporation.
12. The method of claim 1, wherein the non-naturally occurring polynucleotide and the polynucleotide encoding the serine recombinase are contained in a single vector.
13. The method of claim 1, wherein the non-naturally occurring polynucleotide encodes a gene switch system for ligand-inducible control of heterologous gene expression.
14. The method of any one of claims 1-13, wherein the transgene encodes a T-cell receptor.
15. The method of any one of claims 1-13, wherein the transgene encodes a chimeric antigen receptor.
16. The method of claim 15, wherein the wherein the chimeric antigen receptor is capable of binding at least one of CD19, CD33, BCMA, CD44, a-Folate receptor, CAIX, CD30, RORI, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-Al, h5T4, PSMA, TAG-72, EGFRvIII, CD123, and VEGF-R2.
17. The method of claim 15, wherein the chimeric antigen receptor is capable of binding at least one of CD19, CD22, BCMA, EGFRvIII, CD33, ROR, mesothelin, and MUC-16.
18. The method of claim 15, wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 210 or 211.
19. The method of claim 18, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 210 or 211 or a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions.
20. The method of claim 19, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 210 or 211.
21. The method of claim 15, wherein the chimeric antigen receptor comprises: (a) an amino acid sequence having at least 90% identity with SEQ ID NO: 214; and (b) an amino acid sequence having at least 90% identity with SEQ ID NO: 215.
22. The method of claim 21, wherein the chimeric antigen receptor comprises: (a) the amino acid sequence of SEQ ID NO: 214 or a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions; and (b) the amino acid sequence of SEQ ID NO: 215 or a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions.
23. The method of claim 22, wherein the chimeric antigen receptor comprises: (a) the amino acid sequence of SEQ ID NO: 214; and (b) the amino acid sequence of SEQ ID NO: 215.
24. The method of claim 15, wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 216.
25. The method of claim 24, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 216 of a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions.
26. The method of claim 25, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 216.
27. The method of claim 15, wherein the chimeric antigen receptor comprises:

(a) an amino acid sequence having at least 90% identity with SEQ ID NO: 218 and an amino acid sequence having at least 90% identity with SEQ ID NO: 219;

(b) an amino acid sequence having at least 90% identity with SEQ ID NO: 220 and an amino acid sequence having at least 90% identity with SEQ ID NO: 221;

(c) an amino acid sequence having at least 90% identity with SEQ ID NO: 222 and an amino acid sequence having at least 90% identity with SEQ ID NO: 223;

(d) an amino acid sequence having at least 90% identity with SEQ ID NO: 224 and an amino acid sequence having at least 90% identity with SEQ ID NO: 225; or

(e) an amino acid sequence having at least 90% identity with SEQ ID NO: 226 and an amino acid sequence having at least 90% identity with SEQ ID NO: 227.
28. The method of claim 15, wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 228-232.
29. The method of claim 15, wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 233-244.
30. The method of any one of claims 1-13, wherein the non-naturally occurring polynucleotide further encodes a cytokine.
31. The method of claim 30, wherein the cytokine is IL-I, IL-2, IL-12, IL-15, or IL-21, or a functional variant of any one of the foregoing.
32. The method of claim 30, wherein the cytokine is a membrane-bound cytokine.
33. The method of claim 30, wherein the membrane-bound cytokine is membrane-bound IL 15.
34. The method of claim 30, wherein the non-naturally occurring polynucleotide encodes a fusion protein comprising: (a) IL-15, or a functional variant thereof; and (b) IL-15Ra, or a functional variant thereof.
35. The method of claim 30, wherein the non-naturally occurring polynucleotide encodes a fusion protein comprising a sequence having at least 90% identity with SEQ ID NO: 205.
36. The method of claim 35, wherein the non-naturally occurring polynucleotide encodes a fusion protein comprising the sequence of SEQ ID NO: 205 or a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions.
37. The method of claim 36, wherein the non-naturally occurring polynucleotide encodes a fusion protein comprising the sequence of SEQ ID NO: 205.
38. The method of any one of claims 1-13, wherein the non-naturally occurring polynucleotide further encodes a cell tag.
39. The method of claim 38, wherein the cell tag comprises: (a) a truncated HERI, or a functional variant thereof; and/or (b) a truncated CD20, or a functional variant thereof.
40. The method of claim 38, wherein the cell tag comprises an amino acid sequence having at least 90% identity with of any one of SEQ ID NOs: 189-202.
41. The method of claim 38, wherein the cell tag comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 190.
42. The method of claim 41, wherein the cell tag comprises the amino acid sequence of SEQ ID NO: 190 of a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions.
43. The method of claim 42, wherein the cell tag comprises the amino acid sequence of SEQ ID NO: 190.
44. The method of claim 1, wherein the non-naturally occurring polynucleotide encodes: (a) a chimeric antigen receptor; (b) a cytokine; and (c) a cell tag.
45. The method of claim 1, wherein the non-naturally occurring polynucleotide encodes: (a) a chimeric antigen receptor capable of binding at least one of CD19, CD22, BCMA, EGFRvIII, CD33, RORI, mesothelin, and MUC-16; (b) a membrane-bound IL-15; and (c) a truncated HER1, or a functional variant thereof.
46. The method of claim 44, wherein the non-naturally occurring polynucleotide encodes: (a) an amino acid sequence having at least 90% identity with SEQ ID NO: 214; (b) an amino acid sequence having at least 90% identity with SEQ ID NO: 215; (c) an amino acid sequence having at least 90% identity with SEQ ID NO: 205; and (d) an amino acid sequence having at least 90% identity with SEQ ID NO: 190.
47. The method of claim 44, wherein the non-naturally occurring polynucleotide encodes: (a) an amino acid sequence having at least 90% identity with SEQ ID NO: 216; (b) an amino acid sequence having at least 90% identity with SEQ ID NO: 205; and (c) an amino acid sequence having at least 90% identity with SEQ ID NO: 190.
48. The method of claim 47, wherein the non-naturally occurring polynucleotide encodes: (a) the amino acid sequence of SEQ ID NO: 216 or a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions; (b) the amino acid sequence of SEQ ID NO: 205 or a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions; and (c) the amino acid sequence of SEQ ID NO: 190 or a functional variant thereof that differs therefrom by up to 20 conservative amino acid substitutions.
49. The method of claim 48, wherein the non-naturally occurring polynucleotide encodes: (a) the amino acid sequence of SEQ ID NO: 216; (b) the amino acid sequence of SEQ ID NO: 205; and (c) the amino acid sequence of SEQ ID NO: 190.